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Abstract Cushing’s syndrome with concurrent primary aldosteronism (PA) is a rare presentation, and establishing an early diagnosis is imperative to preventing morbidity and long-term sequelae. The diagnosis is established by sequential lab work, showing an elevated cortisol and aldosterone level. Taking the above into consideration, it is evident that repeatedly negative results on all three tests can present an extremely challenging case. In this report, we discuss a female who presented with an adrenal incidentaloma and features suggestive of primary hyperaldosteronism as well as Cushing’s syndrome but no elevations in serum, urine, or salivary cortisol. In this study, we present a 37-year-old female with resistant hypertension and tachycardia. She had several features suggestive of Cushing’s syndrome including resistant hypertension, proximal muscle weakness, weight gain, easy bruising, hair loss, and a history of tachycardia and chest pain. Examination revealed an obese female with thin silvery abdominal striae. The patient’s labs revealed normal serum cortisol, urine-free cortisol (UFC), late-night salivary cortisol, and a normal dexamethasone suppression test. An abdominal computed tomography (CT) scan revealed a right adrenal mass measuring 2.1 x 1.5 x 2.5 cm. Due to a high index of suspicion, adrenal venous sampling was performed, which revealed high levels of cortisol and aldosterone in the right vein, confirming the diagnosis. The patient subsequently underwent a right adrenalectomy. She developed hypotension post-op, leading to the diagnosis of glucocorticoid-remediable aldosteronism. Introduction Primary aldosteronism (PA) is the excess production of aldosterone by the adrenal glands, despite a low serum renin level. The presentation of hyperaldosteronism can be vague and include symptoms such as muscle weakness, fatigue, headaches, numbness, and cramps. More specific findings include resistant hypertension, low serum potassium, and metabolic alkalosis. The etiologies are variable and can include an adrenal adenoma (Conn syndrome) or bilateral adrenal hyperplasia [1]. Cushing’s syndrome is also caused by excess hormone secretion by the adrenal glands. The etiologies include a primary adrenal adenoma, hyperplasia, carcinoma, or exogenous corticosteroid use. It can also be caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma or as a result of paraneoplastic ACTH secretion. The clinical presentation is highly variable and leads to difficulties in establishing a diagnosis. The concurrent existence of primary hyperaldosteronism and Cushing’s syndrome creates additional hindrances in diagnosis, yet further obscured in a patient with a repeatedly negative workup for both conditions. Case Presentation A 37-year-old female presented to her primary care physician with complaints of proximal muscle weakness, tachycardia, and chest pain. Repeated blood pressure readings revealed that she was hypertensive, and she was started on amlodipine and benazepril, which elevated her blood pressure further. A computed tomography (CT) scan (Figure 1) of the abdomen was performed due to resistant hypertension, which revealed an adrenal incidentaloma (right adrenal gland measuring 2.1 x 1.5 x 2.5 cm). Precontract density was 5 Hounsfield units, and a 15-minute delayed washout showed 11 Hounsfield units for a 72% washout. She was thus referred to endocrinology. Figure 1: Abdominal CT scan showing a nodule in the right adrenal gland measuring 2.1 x 1.5 x 2.5 cm She presented to the endocrinology clinic on March 12, 2021. A thorough physical examination was performed, which revealed a well-appearing obese female (BMI of 38.86 kg/m2) with no acute distress. Her blood pressure was 144/108 mmHg, her pulse was 95, and she was afebrile. Thin silvery striations were present on the abdomen, and alopecia was present on the crown. A review of all other systems was unremarkable. A detailed family history revealed early-onset hypertension in her brother (age: 35 years) and her mother (age: 30 years). Personal history included elevated anxiety, weight gain, headaches (frontal band distribution), increased thirst, easy bruising as well as delayed clearance of bruises, and proximal muscle weakness presenting as difficulty in climbing stairs and inability to lift heavy objects. She reported no change in menstrual cycles. There was no history of exogenous corticosteroid use. Serum biochemistries were sent (Table 1), which showed normal levels of thyroid stimulating hormone (TSH), creatinine, liver function tests, and serum electrolytes. However, mildly elevated aldosterone (23 ng/dl), mild hypokalemia (3.3 mEq/L), and suppressed ACTH and dehydroepiandrosterone (DHEA) sulfate were discovered. The aldosterone to renin ratio was also elevated at 59.9 on spironolactone and was 71.4 three months later when spironolactone was discontinued. These findings lead to a preliminary diagnosis of primary hyperaldosteronism. Test Result Calcium 9.1 mmol/L Sodium 137 mmol/L Potassium 4.1 mmol/L Chloride 106 mmol/L CO2 27 BUN 15 mmol/L Glucose 95 mmol/L Creatinine 1.1 μmol/L AST 24 U/L ALT 20 U/L Albumin 4.4 g/L Total protein 7.0 g/L Total bilirubin 0.4 μmol/L Alkaline phosphatase 40 U/L Renin 0.44 Table 1: Patient serum biochemistries BUN: Blood urea nitrogen; AST: Aspartate transaminase; ALT: Alanine transaminase. A workup for elevated cortisol was also performed as the patient was phenotypically Cushingoid, and the following biochemistries were sent sequentially: serum cortisol, 24-hour urine-free cortisol (UFC), salivary cortisol, and a low-dose dexamethasone suppression test (Table 2). The bloodwork was hence nonconfirmatory. Endocrine workup Serum cortisol 4.5 mcg/dL Urine-free cortisol 1.57 g/24 h Salivary cortisol <0.03 μg/dL Dexamethasone suppression test 1.5 mcg/dL Aldosterone <4.0 Table 2: Patient follow-up bloodwork Despite a repeatedly negative workup for Cushing's syndrome, adrenal venous sampling was performed due to a high index of suspicion. The results revealed an inferior vena cava (IVC) cortisol of 20, left adrenal venous (LAV) cortisol of 81, and right adrenal vein (RAV) cortisol of 1280. The results of the IVC aldosterone were 24, LAV aldosterone was 660 and RAV aldosterone was 1500. The elevated levels of cortisol in the RAV were in complete contradiction to the aforementioned workup. A diagnosis of Cushing’s syndrome and concurrent PA was determined. Adrenal veinous sampling was instrumental in establishing the diagnosis but was equivocal and did not lateralize aldosterone and cortisol excess. However, the amount of aldosterone and cortisol were both significantly higher on the right side. After a panel discussion with doctors from several disciplines, a laparoscopic adrenalectomy was planned. The procedure was successful, and the patient was initially showing clinical improvement. The specimen was sent for pathological evaluation and revealed an adrenal cortical adenoma. After initial improvement, the patient developed hypotension, which was likely due to adrenal insufficiency. The patient was supplemented with 1-mg dexamethasone tablets, which stabilized her condition, and a diagnosis of glucocorticoid-remediable-aldosteronism was made. Based on a strong family history of early onset-resistant hypertension, a genetic component was suspected. Several genes associated with PA with autosomal dominant inheritance have been identified [2], such as CYP11B2, CLCN2, KCNJ5, CACNA1D, and CACNA1H. The patient was offered genetic testing but was unable to follow through due to financial reasons. Discussion This patient presented as an extremely rare example of PA and Cushing’s syndrome, with negative serum cortisol, 24-hour UFC, late-night salivary cortisol, and a dexamethasone suppression test. Despite repeatedly negative lab results, the patient presented with a markedly elevated cortisol on adrenal venous sampling. In our literature search, we found an instance of a patient with several negative UFCs [3]; however, to the best of our knowledge, there have been no reported instances of a completely negative workup in a patient who is positive for Cushing’s syndrome. In fact, in the practice guidelines published by the Journal of Clinical Endocrinology & Metabolism [4], it is recommended that patients with a suspected diagnosis of Cushing’s syndrome or an adrenal incidentaloma and two concordant negative test results need not undergo further investigations. One proposed mechanism for the misleading workup could be assay interference. Interference occurs when a substance or process falsely alters an assay result [5]. This can lead to incorrect diagnosis and subsequent treatment and poses a threat to the patient. Another suggested mechanism causing false negative test results could be the hook effect [6]. The hook effect is described as a phenomenon that leads to falsely low results due to the presence of excessive analyte. In a study by Friedman et al. [7], it was noted that patients with “episodic Cushing’s syndrome” or those with mild symptoms had a negative workup. The study recommended serial monitoring for the disease. The interesting fact is that our patient had several features suggestive of active Cushing’s syndrome, and the hypotension seen postoperatively was a testament to the fact that there was in fact a cortisol excess, which led to adrenal insufficiency. In light of the above, a consistently negative workup is perplexing. Zhang et al. suggested performing a low-dose dexamethasone suppression test in individuals presenting with PA, prior to adrenal vein sampling (AVS) and surgery due to the high prevalence of Cushing’s syndrome in patients with PA [8]. A positive test result can lead to a straightforward diagnosis; however, in this rare case where the patient had severe negative tests, it can present as a challenge in diagnosis and treatment. Conclusions The presence of PA and concurrent Cushing’s syndrome can present as a diagnostic challenge. It is recommended to follow up on the signs of Cushing's syndrome with preliminary tests and to presume its absence if two concordant tests are negative. Our patient, however, was an exceptional case. This case highlighted the importance of maintaining a high index of suspicion for patients presenting with several signs and symptoms of the disease and a negative workup. More attention should be paid to the patient's history, and a thorough physical examination should be conducted. In those with an uncertain diagnosis, adrenal venous sampling can provide a clearer picture and lead to a more accurate understanding of the case. References Reincke M, Bancos I, Mulatero P, Scholl UI, Stowasser M, Williams TA: Diagnosis and treatment of primary aldosteronism. Lancet Diabetes Endocrinol. 2021, 9:876-92. 10.1016/S2213-8587(21)00210-2 Dutta RK, Söderkvist P, Gimm O: Genetics of primary hyperaldosteronism. Endocr Relat Cancer. 2016, 23:R437-54. 10.1530/ERC-16-0055 Moloney KJ, Mercado JU, Ludlam WH, Broyles FE: Diagnosis of Cushing's disease in a patient with consistently normal urinary free cortisol levels: a case report. Clin Case Rep. 2016, 4:1181-3. 10.1002/ccr3.647 Nieman LK, Biller BM, Findling JW, Newell-Price J, Savage MO, Stewart PM, Montori VM: The diagnosis of Cushing's syndrome: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2008, 93:1526-40. 10.1210/jc.2008-0125 Dimeski G: Interference testing. Clin Biochem Rev. 2008, 29:S43-8. The hook effect. (2014). Accessed: June 19, 2023: https://www.aacc.org/science-and-research/clinical-chemistry-trainee-council/trainee-council-in-english/pearls-of-lab.... Friedman TC, Ghods DE, Shahinian HK, et al.: High prevalence of normal tests assessing hypercortisolism in subjects with mild and episodic Cushing's syndrome suggests that the paradigm for diagnosis and exclusion of Cushing's syndrome requires multiple testing. Horm Metab Res. 2010, 42:874-81. 10.1055/s-0030-1263128 Zhang Y, Tan J, Yang Q, et al.: Primary aldosteronism concurrent with subclinical Cushing's syndrome: a case report and review of the literature. J Med Case Rep. 2020, 14:32. 10.1186/s13256-020-2353-8 From https://www.cureus.com/articles/170896-rare-challenges-in-diagnosing-cushings-syndrome-and-primary-aldosteronism-a-case-report-of-a-female-with-a-negative-workup#!/

Abstract Background: Cushing syndrome (CS) is a rare disease caused by excess cortisol levels with high cardiovascular morbidity and mortality. Hypertension in CS promotes hypercortisolism-associated cardiovascular events. Adipose tissue is a highly plastic tissue with most cell types strongly affected by the excess cortisol exposure. We hypothesized that the molecular and cellular changes of periadrenal adipose tissue in response to cortisol excess impact systemic blood pressure levels in patients with CS. Methods: We investigated gene expression signatures in periadrenal adipose tissue from patients with adrenal CS collected during adrenal surgery. Results: During active CS we observed a downregulation of gene programs associated with inflammation in periadrenal adipose tissue. In addition, we observed a clustering of the patients based on tissue gene expression profiles into 2 groups according to blood pressure levels (CS low blood pressure and CS high blood pressure). The 2 clusters showed significant differences in gene expression pattens of the renin-angiotensin-aldosterone-system. Renin was the strongest regulated gene compared with control patients and its expression correlated with increased blood pressure observed in our patients with CS. In the CS high blood pressure group, systemic renin plasma levels were suppressed indicative of an abnormal blood pressure associated with periadrenal adipose tissue renin-angiotensin-aldosterone-system activation. Conclusions: Here, we show for the first time a relevant association of the local renin-angiotensin-aldosterone-system and systemic blood pressure levels in patients with CS. Patients from the CS high blood pressure group still had increased blood pressure levels after 6 months in remission, highlighting the importance of local tissue effects on long-term systemic effects observed in CS. Footnotes *U. Stifel and F. Vogel contributed equally. For Sources of Funding and Disclosures, see page xxx. Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/HYPERTENSIONAHA.123.21185. Correspondence to: Martin Reincke, Department of Medicine IV, University Hospital, LMU Munich, GermanyEmail martin.reincke@med.uni-muenchen.de Jan Tuckermann, Institute of Comparative Molecular Endocrinology (CME), Ulm University, GermanyEmail jan.tuckermann@uni-ulm.de eLetters eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate. Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page. From https://www.ahajournals.org/doi/10.1161/HYPERTENSIONAHA.123.21185

Objective: To evaluate the long-term efficacy and safety of osilodrostat in patients with Cushing’s disease. Methods: The multicenter, 48-week, Phase III LINC 4 clinical trial had an optional extension period that was initially intended to continue to week 96. Patients could continue in the extension until a managed-access program or alternative treatment became available locally, or until a protocol amendment was approved at their site that specified that patients should come for an end-of-treatment visit within 4 weeks or by week 96, whichever occurred first. Study outcomes assessed in the extension included: mean urinary free cortisol (mUFC) response rates; changes in mUFC, serum cortisol and late-night salivary cortisol (LNSC); changes in cardiovascular and metabolic-related parameters; blood pressure, waist circumference and weight; changes in physical manifestations of Cushing’s disease; changes in patient-reported outcomes for health-related quality of life; changes in tumor volume; and adverse events. Results were analyzed descriptively; no formal statistical testing was performed. Results: Of 60 patients who entered, 53 completed the extension, with 29 patients receiving osilodrostat for more than 96 weeks (median osilodrostat duration: 87.1 weeks). The proportion of patients with normalized mUFC observed in the core period was maintained throughout the extension. At their end-of-trial visit, 72.4% of patients had achieved normal mUFC. Substantial reductions in serum cortisol and LNSC were also observed. Improvements in most cardiovascular and metabolic-related parameters, as well as physical manifestations of Cushing’s disease, observed in the core period were maintained or continued to improve in the extension. Osilodrostat was generally well tolerated; the safety profile was consistent with previous reports. Conclusion: Osilodrostat provided long-term control of cortisol secretion that was associated with sustained improvements in clinical signs and physical manifestations of hypercortisolism. Osilodrostat is an effective long-term treatment for patients with Cushing’s disease. Clinical trial registration: ClinicalTrials.gov, identifier NCT02180217 Introduction Cushing’s disease is a rare but serious disorder resulting from an adrenocorticotropic hormone (ACTH)-producing pituitary adenoma that, in turn, promotes excess adrenal cortisol (1). Chronic exposure to excess cortisol is associated with numerous comorbidities, including hypertension, muscle weakness, hirsutism, central obesity, hypercoagulability and diabetes mellitus, all of which lead to an increased risk of mortality and poor health-related quality of life (HRQoL) (1–3). The longer the exposure to excess cortisol, the lower the chance of reversing morbidity (2). Although transsphenoidal surgery is the recommended first-line treatment, approximately one-third of patients experience persistent or recurrent disease following surgery (4), and some patients are ineligible for or refuse surgery (4–6). Steroidogenesis inhibitors are usually the first choice for medical treatment (6). The effect of medical treatment can be easily monitored by measurement of serum and urine cortisol. Owing to the unremitting nature of Cushing’s disease, patients often require continued medical therapy to maintain long-term control of cortisol excretion. To date, long-term efficacy and safety data for steroidogenesis inhibitors from prospective clinical trials are limited (7, 8). Osilodrostat is a potent oral inhibitor of 11β-hydroxylase and is approved for the treatment of adult patients with Cushing’s disease (USA) or endogenous Cushing’s syndrome (EU and Japan) who are eligible for medical therapy (9–12). The LINC 4 study was a multicenter, 48-week, Phase III clinical trial in patients with Cushing’s disease that included an upfront 12-week randomized, double-blind, placebo-controlled period. Osilodrostat led to rapid normalization of mean urinary free cortisol (mUFC) excretion and was significantly superior to placebo at week 12; normal mUFC excretion was sustained in most patients throughout the 48-week core period (13). Following the 48-week core period, patients could enter an optional open-label extension period intended to run for an additional 48 weeks. Here, we report the long-term efficacy and safety data from the extension of LINC 4. These data augment the existing efficacy and safety profile of osilodrostat (7, 8, 13, 14). Methods Patients Eligibility criteria have been described previously (13). Briefly, the study enrolled adult patients with a confirmed diagnosis of persistent or recurrent Cushing’s disease after pituitary surgery and/or irradiation, or de novo Cushing’s disease (if not surgical candidates), with mUFC >1.3 times the upper limit of normal (ULN; 138 nmol/24 h or 50 μg/24 h; calculated from three samples collected on three consecutive days, with ≥2 values >1.3 x ULN). Patients who continued to receive clinical benefit from osilodrostat, as assessed by the study investigator, could enter the extension phase. The study was conducted in accordance with the Declaration of Helsinki, with an independent ethics committee/institutional review board at each site approving the study protocol; patients provided written informed consent to participate and consented again at week 48 to taking part in the extension phase. The trial is registered at ClinicalTrials.gov (NCT02180217). Study design Data from the 48-week core period of this Phase III study, consisting of a 12-week randomized, placebo-controlled, double-blind period followed by a 36-week open-label treatment period, have been published previously (13). The optional open-label extension phase was initially planned to run for an additional 48 weeks (to week 96 for the last patient enrolled). However, patients could continue in the extension only until a managed-access program or alternative treatment became available locally, or until a protocol amendment was approved at their site that specified that patients enrolled in the optional extension phase should come for an end-of-treatment (EOT) visit within 4 weeks or by week 96, whichever occurred first. Patients still receiving clinical benefit from osilodrostat at their EOT visit were eligible to join a separate long-term safety follow-up study (NCT03606408). Consequently, the extension phase ended when all patients had transitioned to the long-term safety follow-up study, if eligible, or had discontinued from the study. Patients continued to receive open-label osilodrostat at the established effective dose from the core phase (dose adjustments were permitted based on efficacy and tolerability; the maximum dose was 30 mg twice daily [bid]). Outcomes Study outcomes assessed during the extension phase were as follows: complete (mUFC ≤ULN), partial (mUFC decrease ≥50% from baseline and >ULN) and mUFC response rate at weeks 60, 72, 84, 96 and 108, then every 24 weeks until the extension EOT visit; change in mUFC, serum cortisol and late-night salivary cortisol (LNSC) at weeks 60, 72, 84, 96 and 108, then every 24 weeks until the extension EOT visit; time to loss of mUFC control, defined as the time (in weeks) from the first collection of post-baseline normal mUFC (≤ULN) to the first mUFC >1.3 x ULN on two consecutive scheduled visits on the highest tolerated dose of osilodrostat and not related to a dose interruption or reduction for safety reasons after week 26; change in cardiovascular/metabolic-related parameters associated with Cushing’s disease (fasting plasma glucose [FPG] and glycated hemoglobin [HbA1c]) at weeks 60, 72, 84, 96 and 108, then every 24 weeks until the extension EOT visit; blood pressure, waist circumference and weight every 4 weeks until week 72, then every 12 weeks until week 108, then every 24 weeks until the extension EOT visit; change from baseline in physical manifestations of hypercortisolism at weeks 72, 96 and 108, then every 24 weeks until the extension EOT visit; changes in HRQoL (determined by Cushing’s Quality of Life Questionnaire [CushingQoL] and Beck Depression Inventory II [BDI-II]) at weeks 72 and 96 and the extension EOT visit; and proportion of patients with ≥20% decrease or increase in tumor volume. mUFC (mean of two or three 24-hour urine samples), serum cortisol (measured between 08:00 and 10:00) and LNSC (measured from two samples collected between 22:00 and 23:00) were evaluated using liquid chromatography-tandem mass spectrometry and assessed centrally. Pituitary magnetic resonance imaging with and without gadolinium enhancement was performed locally at weeks 72 and 96 and the extension EOT visit; images were assessed centrally for change in tumor size. Safety was continually assessed from core study baseline throughout the extension for all enrolled patients by monitoring for adverse events (AEs); all AEs from first patient first visit to last patient last visit are reported. AEs of special interest (AESIs) included events related to hypocortisolism, accumulation of adrenal hormone precursors, arrhythmogenic potential and QT prolongation, and enlargement of the pituitary tumor. Statistical methods Analyses presented here are based on cumulative data generated for the full analysis set (all patients enrolled at core study start who received at least one dose of osilodrostat) up to last patient last visit. Safety analyses included all enrolled patients who received at least one dose of osilodrostat and had at least one valid post-baseline safety assessment. All analyses excluded data for patients in the placebo arm collected during the placebo-controlled period. Results were analyzed descriptively, and no formal statistical testing was performed. Correlations were evaluated using the Pearson’s correlation coefficient; extreme outliers were defined as >(Q3 + 3 x IQR) or <(Q1 − 3 x IQR), where Q1 and Q3 are the first and third quartiles and IQR is the interquartile range (Q3 − Q1). Results Patient disposition and baseline characteristics LINC 4 was conducted from October 3, 2016 to December 31, 2020. Of the 73 patients who were enrolled and received treatment in the core phase, 65 completed the core phase and 60 (82.2%) opted to enter the extension; 53 (72.6%) patients completed the extension (Figure 1). At core study baseline, most patients had undergone previous pituitary surgery (87.7%) or received prior medical therapy (61.6%; Table 1). Patients had a variety of comorbidities at core study baseline, most commonly hypertension (61.6%); physical manifestations of hypercortisolism were common (Table 1). Figure 1 Figure 1 Patient disposition. *Patient was randomly allocated to osilodrostat but did not receive any study treatment because of a serious AE (grade 4 pituitary apoplexy that required hospitalization prior to receiving any study drug) that was not considered related to treatment. Table 1 Table 1 Core study patient baseline characteristics. Exposure to osilodrostat From core baseline to study end, median (range) osilodrostat exposure was 87.1 (2.0–126.6) weeks; 29 (39.7%) patients were exposed to osilodrostat for more than 96 weeks. The median (25th–75th percentiles) average osilodrostat dose received during the overall study period was 4.6 (3.7–9.2) mg/day; during the core study, median (25th–75th percentiles) average dose was 5.0 (3.8–9.2) mg/day (13). The osilodrostat dose being taken for the longest duration was most frequently 4.0 mg/day (27.4%). Following titration, daily osilodrostat dose remained stable during long-term treatment (Figure 2). Figure 2 Figure 2 (A) Mean and (B) median osilodrostat dose over time. Shaded areas indicate the randomized, double-blind period and the open-label period of the core phase. According to the study protocol, all patients restarted the open-label period on osilodrostat 2 mg bid unless they were on a lower dose at week 12. All patients on <2 mg bid osilodrostat (or matched placebo) at week 12 continued to receive the same dose, regardless of initial treatment allocation. n is the number of patients who contributed to the mean/median. Long-term efficacy of osilodrostat treatment Of patients who had received at least one dose of osilodrostat, 68.5% (n=50/73) had mUFC ≤ULN at the end of the core period, and 54.8% (n=40/73) had mUFC ≤ULN at week 72. Of patients who opted to enter the extension, 66.7% had mUFC ≤ULN (n=40/60) and 8.3% (n=5/60) had mUFC decreased by ≥50% from baseline and >ULN at week 72 (Figure 3A). Of patients with an assessment at their extension EOT visit, 72.4% (n=42/58) had mUFC ≤ULN and 8.6% (n=5/58) had mUFC decreased by ≥50% from baseline and >ULN. Figure 3 Figure 3 (A) Proportion of patients with mUFC response over time, (B) mean mUFC over time, and (C) individual patient changes in mUFC. (A) Patients with missing mUFC at any visit, including those who had discontinued treatment, were counted as non-responders. Shaded area represents the 48-week core phase; excludes data in placebo arm collected during placebo-control period. *The proportion of patients with mUFC ≤ULN at week 48 was calculated using the full analysis set (patients who had discontinued treatment were classified as non-responders). †Discontinued, n=12; missing because of the COVID-19 pandemic, n=4; mUFC not meeting response criteria, n=3; missing (any other reason), n=1. ‡mUFC not meeting response criteria, n=8; missing because of the COVID-19 pandemic, n=2; missing (any other reason), n=1. (B) Shaded areas indicate the randomized, double-blind period and the open-label period of the core phase. n is the number of patients who contributed to the mean. Analysis includes scheduled visits only. (B, C) Dashed line is the ULN for UFC (138 nmol/24 h). Mean mUFC excretion for the 48-week core period of the study has been reported previously (13); mUFC excretion normalized in patients who received osilodrostat, either during the 12-week randomized period (osilodrostat arm) or during the subsequent 36-week open-label period (all patients) (13). Mean mUFC excretion was maintained within the normal range in the extension period (week 72 (n=48), 90.5 [SD 122.6] nmol/24 h; 0.7 [0.9] x ULN; Figure 3B). Median (range) mUFC excretion is shown in Supplementary Figure 1A. Individual patient changes in mUFC from core study baseline to their last observed visit are shown in Figure 3C. There were no escape-from-response events during the extension phase following the primary analysis cut-off (February 25, 2020) (13). During the core period, mean (SD) serum cortisol levels decreased from 538.1 (182.3) nmol/L (0.9 [0.3] x ULN) at baseline to 353.9 (124.9) nmol/L (0.6 [0.2] x ULN) at week 48. Serum cortisol levels then remained stable throughout the extension period (week 72: 319.1 [129.8] nmol/L, 0.6 [0.2] x ULN; Figure 4A). LNSC also decreased and then remained stable, although >ULN, throughout the study (baseline: 10.8 [23.5] nmol/L, 4.3 [9.4] x ULN; week 48: 3.7 [2.6] nmol/L, 1.5 [1.0] x ULN; week 72: 3.8 [3.0] nmol/L, 1.5 [1.2] x ULN; Figure 4B). Median serum cortisol and LNSC are shown in Supplementary Figures 1B, C. Of patients with baseline and last observed value (LOV) measurements, 25.0% had normal LNSC at baseline (n=6/24) and 47.8% had normal LNSC at their last visit (n=11/23). Interpretation of this result is limited by the high degree of missing data (baseline: 67.1%, n=49/73; LOV: 68.5%, n=50/73). Figure 4 Figure 4 (A) Mean serum cortisol and (B) mean LNSC from baseline to the end of treatment. Shaded areas indicate the randomized, double-blind period and the open-label period of the core phase. n is the number of patients who contributed to the mean. Dashed line in (A) indicates reference serum cortisol range for males and females ≥18 years old (127–567 nmol/L). Dashed line in (B) indicates reference LNSC (22:00–23:00) range for males and females ≥18 years old (≤2.5 nmol/L). Changes in cardiovascular and metabolic parameters, physical manifestations of Cushing’s disease and patient-reported outcomes As previously reported, improvements from baseline occurred in most cardiovascular and metabolic-related parameters in the core period following osilodrostat treatment (9). This trend continued during the extension phase and included a reduction in FPG, HbA1c, cholesterol, systolic and diastolic blood pressure, waist circumference, and weight (Figure 5). Similarly, the improvements from baseline in physical features of hypercortisolism observed by week 48 were maintained for most parameters throughout the extension (Figure 6A), with either no change or improvement observed from baseline in ≥90% patients for all parameters at week 72. Facial rubor, supraclavicular fat pad, dorsal fat pad and central obesity had a favorable shift from baseline in ≥40% of patients at week 72. Few patients reported worsening from baseline of specific manifestations (Figure 6A). Figure 5 Figure 5 Changes in cardiovascular-related metabolic parameters. Shaded area indicates the core phase. n is the number of patients who contributed to the mean. Error bars indicate standard deviation. DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SBP, systolic blood pressure. Figure 6 Figure 6 Changes in (A) physical manifestations of Cushing’s disease and (B) patient-reported outcomes. Shaded area indicates the core phase. n is the number of patients who contributed to the mean. Improvements were also observed in scores for patient quality of life (QoL). Both standardized CushingQoL and BDI-II scores improved steadily during the core phase. QoL scores continued to improve further during the extension. At week 72 and EOT, mean (SD) standardized CushingQoL score was 66.4 (19.6) and 69.0 (20.9), and mean (SD) BDI-II score was 6.5 (7.0) and 6.2 (7.1), representing a mean (SD) change from baseline of 15.2 (19.0) and 17.1 (17.1) and −4.1 (9.3) and −4.5 (7.9), respectively (Figure 6B). Adverse events AEs that occurred in >20% of patients, irrespective of study-drug relationship, during the entire study period (median [range] osilodrostat exposure for all patients: 87.1 [2.0–126.6] weeks; excluding data collected in the placebo arm during the placebo-controlled period) are shown in Table 2. The most common AEs were decreased appetite (46.6%), arthralgia (45.2%) and fatigue (39.7%). Most AEs were mild or moderate; 60.3% were reported as grade 1/2 (Table 2). Table 2 Table 2 Summary of adverse events during LINC 4 core and extension periods. Overall, 10 AEs (adrenal insufficiency, n=3; hyperbilirubinemia, hypokalemia, headache, arthralgia, pituitary tumor, benign pituitary tumor, and depression, n=1 each) in nine patients (12.3%; one patient experienced both arthralgia and headache) led to treatment discontinuation. For two patients (2.7%), those AEs were reported as grade 3 (hyperbilirubinemia and hypokalemia). One patient discontinued following the primary analysis cut-off date (February 25, 2020). The most common AESIs in both the core and extension periods were those related to adrenal hormone precursors. However, the proportion of patients reporting these AESIs was lower in the extension than in the core period (Figure 7). AESIs related to hypocortisolism were most frequent during the core period but did occur throughout the remainder of the study, albeit at lower frequency (Figure 7). Hypocortisolism-related AEs were most frequently managed with temporary osilodrostat interruption (n=20) or dose adjustment (n=6), and with concomitant glucocorticoids (n=15). There were no new occurrences of AESIs related to arrhythmogenic potential and QT prolongation, or to pituitary tumor enlargement, in the extension (Figure 7). During the entire study period from core baseline to the end of the extension, AESIs led to osilodrostat discontinuation in six (8.2%) patients (n=1, related to accumulation of adrenal hormone precursors [hypokalemia]; n=3, related to hypocortisolism [all adrenal insufficiency]; n=2, related to pituitary tumor enlargement [pituitary tumor and pituitary tumor benign]). Figure 7 Figure 7 Occurrence of AESIs by time interval. The denominator for each time period only included patients who had at least one scheduled visit, or at least one observed AE, during that period. From baseline to week 12, the denominator only included patients randomized to osilodrostat. A patient with multiple occurrences of an AE within the same period is counted only once in that period. However, if an AE ends and occurs again in a different period, it is then counted in both periods. Shaded areas indicate the randomized, double-blind period and the open-label period of the core phase. *Maximum duration of follow-up was 127 weeks. Following an increase in 11-deoxycortisol and 11-deoxycorticosterone during the core study, levels tended to decrease during longer-term treatment (Figure 8). From baseline to LOV, the proportion of patients with elevated 11-deoxycorticosterone and 11-deoxycortisol levels increased from 10.0% (n=1/10) to 90.0% (n=9/10) and from 57.9% (n=33/57) to 86.7% (n=5 and 2/60), respectively. In female patients, mean (SD) testosterone levels increased from 1.1 (0.6) nmol/L at baseline to 2.5 (2.6) nmol/L at the end of the core phase, then decreased to within the normal range (0.7−2.6 nmol/L for females) by the extension phase end-of-treatment visit (1.9 [1.7] nmol/L; Figure 8). The proportion of females with an elevated testosterone level increased from 15.0% (n=9/61) at baseline to 63.2% (n=24/61) at week 72 and then reduced to 41.7% (n=25/61) at LOV. In males, testosterone levels increased and remained within the normal range throughout osilodrostat treatment (Figure 8). The proportion of male patients with testosterone levels below the lower limit of normal decreased from 58.3% (n=7/12) at baseline to 33.3% (n=4/12) at LOV. The proportion of patients experiencing AEs potentially related to increased testosterone (increased blood testosterone, acne and hirsutism) was lower during the extension than during the core study (Supplementary Figure 2). Mean serum potassium levels remained stable and within the normal range (3.5–5.3 mmol/L) throughout osilodrostat treatment (Figure 8). The proportion of patients with a normal potassium level was similar between baseline (98.6%, n=72/73) and LOV (94.4%, n=68/72). Figure 8 Figure 8 Mean (± SD) levels up to the end-of-treatment visit in the extension phase for 11-deoxycortisol, 11-deoxycorticosterone, potassium and testosterone (in males and females). Shaded area indicates the core phase. n is the number of patients who contributed to the mean. Reference ranges: 11-deoxycortisol ULN, 3.92 nmol/L in males and 3.1 nmol/L in females, or lower depending on age; 11-deoxycorticosterone ULN, 455 pmol/L in males and 696 pmol/L in females (mid-cycle); potassium, 3.5–5.3 mmol/L; testosterone, 8.4–28.7 nmol/L in males and 0.7–2.6 nmol/L in females. At baseline, median (range) tumor volume was 82.0 (12.0–2861.0) mm3; 28.8% (n=21/73) of patients had a macroadenoma (≥10 mm) and 68.5% (n=51/73) had a microadenoma (<10 mm). At week 72, median (range) tumor volume was 68.0 (10.0–3638.0) mm3 (Figure 9A). Of the 27 patients with measurements at both baseline and week 72, 29.6% (n=8/27) had a ≥20% decrease in tumor volume and 37.0% (n=10/27) had a ≥20% increase (Figure 9B). Notably, mean (SD) plasma ACTH increased steadily between baseline (17.1 [32.1] pmol/L, n=73) and week 72 (65.0 [96.9] pmol/L, n=45; Figure 9C); mean ACTH levels appeared to stabilize after week 72. All patients experienced an increase in ACTH levels from baseline to week 72 (n=45) and LOV (n=73); of these, 34/45 (75.6%) and 47/73 (64.4%) experienced an increase in ACTH of ≥2 × baseline levels to week 72 and to LOV, respectively. There was no correlation between change in tumor volume and change in ACTH from baseline to week 72 (r=0.1; calculated without two extreme outliers). Figure 9 Figure 9 (A) Mean and median tumor volume over time, (B) number of patients with a change in tumor volume from baseline, and (C) mean ACTH over time. Shaded areas indicate the core phase. n is the number of patients who contributed to the mean. Dashed lines in (C) indicate reference morning (07:00–10:00) plasma ACTH ranges for males and females ≥18 years old (1.3–11.1 pmol/L). Discussion Following transsphenoidal surgery, approximately one-third of patients experience persistence or recurrence of disease and subsequently require further treatment to control excess cortisol secretion (4). It is therefore essential that clinical studies evaluating the long-term safety and efficacy of potential new treatments, such as osilodrostat, are performed. The data presented here from the LINC 4 extension reinforce previous reports demonstrating that osilodrostat is effective and well tolerated during long-term treatment of Cushing’s disease (7, 8, 13, 14). The normalization of mUFC excretion, observed from as early as week 2 in some patients (13), was sustained to the end of the optional open-label extension phase. Overall, the response rate was durable and remained ≥60% throughout the study, with 72.4% of patients maintaining mUFC ≤ULN at their extension EOT visit. Considering the range in baseline mUFC values (21.4–2607.3 nmol/24 h), this indicates that patients can benefit from osilodrostat treatment regardless of their baseline mUFC level. This also suggests that baseline mUFC is not an indicator of whether a patient will respond to osilodrostat treatment. Notably, there were no escape events during the extension period. Additionally, the improvements in most cardiovascular and metabolic parameters, physical manifestations and QoL previously reported during the 48-week core phase were maintained or further improved with long-term treatment (13). Collectively, these results demonstrate the ability of osilodrostat to reduce the burden of disease and comorbidities frequently experienced by patients with Cushing’s disease. mUFC excretion is commonly assessed in clinical trials and during routine clinical practice to evaluate response to treatment. It is also important to monitor the recovery of the circadian cortisol rhythm in response to treatment by measuring serum cortisol and LNSC (6, 15–17). Elevated LNSC levels have been linked to dysregulation in glucose tolerance, insulin sensitivity and insulin secretion (18). As such, one potential explanation for persistent comorbidities in some patients with normalized mUFC excretion is that LNSC, although reduced, remains just above the ULN. Assessment of LNSC during treatment with other medical therapies has been reported, although differences in treatment duration and patient population type and size limit meaningful comparisons between therapies (15–17). In LINC 4, mean serum cortisol levels remained within the normal range. Mean LNSC improved considerably from baseline but remained above the ULN throughout the study; 47.8% (n=11/23) of patients achieved normalized LNSC at their LOV visit. A numerically large decrease in LNSC, but with mean levels remaining above the ULN, is consistent with previous reports during long-term osilodrostat treatment (8); the mechanism underlying this observation is currently unknown. In real-life clinical practice, the osilodrostat label allows flexible dosing (9, 11), which may help achieve normalization of LNSC. Furthermore, the number of patients with available LNSC assessments was limited, particularly during the extension; therefore, the data should be interpreted with caution. Future studies should examine whether patients with normalization of both UFC and LNSC have better outcomes than patients with only normalized UFC. Overall, the safety findings reported here for the extension period were consistent with those reported in the primary analysis (13) and previous clinical trials (7, 8, 14). Osilodrostat was generally well tolerated throughout the study; most reported AEs were mild or moderate in severity and manageable. Only nine of 73 (12.3%) patients discontinued osilodrostat at any time because of an AE (3/73 [4.1%] prior to week 48; 6/60 [10.0%] after week 48). Given that osilodrostat is a potent inhibitor of 11β-hydroxylase, AEs related to hypocortisolism or increased levels of adrenal hormone precursors are expected. The frequency of these AEs was lower in the extension period than in the core period, although events did still occur, highlighting the importance of monitoring patients regularly throughout long-term osilodrostat use. AEs potentially related to arrhythmogenic potential and QT prolongation remained infrequent throughout the study. Furthermore, the clinical benefit and tolerability of osilodrostat is supported by the high proportion of patients who chose to continue into the extension period: 92.3% who completed the core phase continued into the optional extension phase, with 88.3% of those completing the extension. Although dose adjustments were allowed in the open-label phase, the dose of osilodrostat remained stable over long-term treatment, with 4 mg/day adequate for most patients to achieve and sustain control of mUFC excretion. Most AEs related to hypocortisolism occurred during the dose-escalation periods of both LINC 4 (27%) and LINC 3 (51%) (19); the lower occurrence in LINC 4 than LINC 3 may have been related to the more gradual dose-escalation schedule of LINC 4 (every 3 weeks) relative to that of LINC 3 (every 2 weeks) (13, 14, 19). As such, an increased dose-titration interval could be considered when there is a need to mitigate the potential for glucocorticoid withdrawal syndrome or hypocortisolism-related AEs following a rapid decrease in cortisol. Dose-increase decisions should be informed by regular cortisol assessments, the rate of decrease in cortisol, and the individual’s clinical response and tolerability to osilodrostat. Furthermore, as with all steroidogenesis inhibitors, patients should be educated on the expected effects of treatment and dose increases, with a particular focus on the symptoms of hypocortisolism and the advice to contact their physician if they occur. As expected, levels of 11-deoxycortisol, 11-deoxycorticosterone and, in women, testosterone increased during osilodrostat treatment. These then decreased during long-term treatment; notably, testosterone levels in women returned to within the normal range and to near baseline levels. These observations are consistent with the findings of LINC 3, which also demonstrated that these increases were reversible following discontinuation of osilodrostat (14). Compared with the primary analysis, there were no new AEs of increased testosterone in the extension phase of LINC 4; these findings are consistent with both LINC 2 and LINC 3 long-term analyses (7, 8). In general, osilodrostat did not adversely affect pituitary tumor volume, with similar proportions of patients reporting either a ≥20% decrease, ≥20% increase or stable tumor volume throughout the study. Although ACTH levels increased during osilodrostat treatment, there was no apparent correlation between the change in ACTH and the change in tumor volume after 72 weeks of treatment; however, longer-term data are needed to evaluate this further. As ACTH-producing pituitary adenomas are the underlying drivers of hypercortisolism, in turn responsible for the high morbidity and poor QoL associated with the disease, tumor stability is of great clinical importance in patients with Cushing’s disease, especially those for whom surgery has failed or is not a viable option. In addition to LINC 4, other studies have assessed the long-term efficacy and safety of other medical therapies (20–24); however, there is a paucity of prospective, long-term data. For metyrapone, an oral steroidogenesis inhibitor that is given three or four times daily (25), prospective data are currently only available for 36 weeks of treatment in the Phase III/IV PROMPT study (22, 23). Normalization of mUFC excretion was observed in 48.6% (n=17/35) of patients at week 36 (23), and gastrointestinal, fatigue and adrenal insufficiency AEs were the most commonly reported during the first 12 weeks of treatment (22). Current data for levoketoconazole, an oral steroidogenesis inhibitor that is a ketoconazole stereoisomer taken twice daily, are available for 12 months (median duration of exposure 15 months, n=60) following the extended open-label extension of the Phase III SONICS study (26). Of patients with data, 40.9% (n=18/44) had normal mUFC excretion at month 12 (26). During the extension, no patient experienced alanine aminotransferase or aspartate aminotransferase >3 x ULN, suggesting that the potentially clinically important events relating to liver toxicity may be more likely to occur early during treatment, although periodic monitoring during long-term treatment is advisable (26). Pasireotide is a second-generation somatostatin receptor ligand that is administered subcutaneously twice daily (27, 28) or intramuscularly once a month (29–31). In a 12-­month extension of a Phase III study evaluating the long-term efficacy of long-acting pasireotide, 53.1% of patients had normalized mUFC at study completion (median treatment duration 23.9 months), with the most common AEs being related to hyperglycemia (21). The differences in duration and design of these studies prevent a meaningful comparison of the long-term efficacy of medical treatments for Cushing’s disease. The extension period of LINC 4 was initially planned to run to week 96; however, in agreement with the FDA, a protocol amendment was approved that resulted in approximately half of the patients completing the extension phase between weeks 72 and 96. We also acknowledge the potential for selection bias for patients who experienced the greatest clinical benefit during the 48-week core study; however, over 80% of patients chose to continue osilodrostat treatment after consenting to take part in the extension. Conclusions During the LINC 4 extension period, osilodrostat provided long-term control of cortisol excretion, accompanied by sustained improvements in clinical symptoms, physical manifestations of hypercortisolism and QoL. The safety profile was favorable. These data provide further evidence of the durable clinical benefit of long-term osilodrostat treatment in patients with persistent, recurrent or de novo Cushing’s disease. Data availability statement The datasets generated and analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request. Recordati Rare Diseases will share the complete de-identified patient dataset, study protocol, statistical analysis plan, and informed consent form upon request, effective immediately following publication, with no end date. Ethics statement The studies involving human participants were reviewed and approved by an independent ethics committee/institutional review board at each study site. The patients/participants provided their written informed consent to participate in this study. Author contributions The study steering committee (PS, AH, RF, and RA), AP, and the funder designed the study. AH, MG, MB, PW, ZB, AT, and PS enrolled patients in the study. Data were collected by investigators of the LINC 4 Study Group using the funder’s data management systems. MP and the funder’s statistical team analyzed the data. A data-sharing and kick-off meeting was held with all authors and an outline prepared by a professional medical writer based on interpretation provided by the authors. Each new draft of the manuscript subsequently prepared by the medical writer was reviewed and revised in line with direction and feedback from all authors. All authors contributed to the article and approved the submitted version. Funding This study was funded by Novartis Pharma AG; however, on July 12, 2019, osilodrostat became an asset of Recordati. Financial support for medical editorial assistance was provided by Recordati. Acknowledgments We thank all the investigators, nurses, study coordinators and patients who participated in the trial. We thank Catherine Risebro, PhD of Mudskipper Business Ltd for medical editorial assistance with this manuscript. Conflict of interest Author MG has received speaker fees from Recordati, Ipsen, Crinetics Pharmaceuticals, and Novo Nordisk and attended advisory boards for Novo Nordisk, Recordati, Ipsen, and Crinetics Pharmaceuticals. Author PS reports consultancy for Teva Pharmaceuticals. Author PW reports receiving travel grants and speaker fees from Novartis, Ipsen, Recordati, Novo Nordisk, Strongbridge Biopharma now Xeris Pharmaceuticals, and Lilly. Author MB reports receiving travel grants from Novartis, Ipsen, and Pfizer and consultancy for Novartis. Author ZB has nothing to disclose. Author AT reports consultancy for CinCor and PhaseBio. Author RF reports consultancy for HRA Pharma and Recordati and a research grant from Corcept Therapeutics. Author AH reports speaker fees from Chiasma and Ipsen and has been an advisor to Strongbridge Biopharma now Xeris Pharmaceuticals, Novo Nordisk, and Lundbeck Pharma. Author MP is employed by the company Novartis Pharma AG. Author AP was employed by the company Recordati AG at the time of manuscript development. Author RA reports grants and personal fees from Xeris Pharmaceuticals, Spruce Biosciences, Neurocrine Biosciences, Corcept Therapeutics, Diurnal Ltd, Sparrow Pharmaceuticals, and Novartis and personal fees from Adrenas Therapeutics, Janssen Pharmaceuticals, Quest Diagnostics, Crinetics Pharmaceuticals, PhaseBio Pharmaceuticals, H Lundbeck A/S, Novo Nordisk, and Recordati Rare Diseases. 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Efficacy and safety of once-monthly pasireotide in Cushing's disease: a 12 month clinical trial. Lancet Diabetes Endocrinol (2018) 6:17–26. doi: 10.1016/S2213-8587(17)30326-1 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: Cushing’s disease, osilodrostat, hypercortisolism, 11β-hydroxylase, long-term treatment Citation: Gadelha M, Snyder PJ, Witek P, Bex M, Belaya Z, Turcu AF, Feelders RA, Heaney AP, Paul M, Pedroncelli AM and Auchus RJ (2023) Long-term efficacy and safety of osilodrostat in patients with Cushing’s disease: results from the LINC 4 study extension. Front. Endocrinol. 14:1236465. doi: 10.3389/fendo.2023.1236465 Received: 07 June 2023; Accepted: 28 July 2023; Published: 23 August 2023. Edited by: Fabienne Langlois, Centre Hospitalier Universitaire de Sherbrooke, Canada Reviewed by: Filippo Ceccato, University of Padua, Italy Kevin Choong Ji Yuen, Barrow Neurological Institute (BNI), United States Copyright © 2023 Gadelha, Snyder, Witek, Bex, Belaya, Turcu, Feelders, Heaney, Paul, Pedroncelli and Auchus. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Mônica Gadelha, mgadelha@hucff.ufrj.br †Present address: Alberto M. Pedroncelli, Camurus AB, Lund, Sweden Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fendo.2023.1236465/full

October 7 @ 9:00 am – 1:00 pm The annual Pituitary Patient Education Day is a free event that features presentations from Johns Hopkins pituitary experts. To RSVP, please email pituitaryday@jhmi.edu. Space is limited. One member per family is encouraged to attend. Presentations Pituitary lesions: acromegaly, Cushing, prolactinomas, non-functioning masses and hypophysitis (Roberto Salvatori, M.D.) How pituitary tumors can affect your vision (Amanda Henderson, M.D.) Medications for pituitary disease: what you should know (Amir Hamrahian, M.D.) The nose as the door to the pituitary gland (Masaru Ishii, M.D., Ph.D.) Surgery for small and large pituitary tumors: images from the Johns Hopkins operating room (Gary Gallia, M.D., Ph.D.) Radiation: when is it needed and how (Lawrence Kleinberg, M.D.) Chevy Chase Auditorium 1800 Orleans Street Baltimore, Maryland 21287+ Google Map More info at https://events.hopkinsmedicine.org/event/johns-hopkins-pituitary-patient-education-day/

Key takeaways: Cushing’s syndrome symptoms moderately impact quality of life for adults with the condition. Weight gain, muscle fatigue and menstrual changes decline in severity from diagnosis to follow-up. Adults with endogenous Cushing’s syndrome reported that the condition moderately affects their quality of life and causes them to have symptoms about 16 days in a given month, according to findings published in Pituitary. “Our study aimed to evaluate the ongoing burden of Cushing’s syndrome in order to identify areas of unmet need,” Eliza B. Geer, MD, medical director of the Multidisciplinary Pituitary and Skull Base Tumor Center and associate attending of endocrinology and neurosurgery at Memorial Sloan Kettering Cancer Center, told Healio. “We found that patients with treated Cushing’s continue to experience ongoing symptoms more than half of the days in a given month, miss about 25 workdays per year and need twice the average number of outpatient visits per year, indicating a significant impact on daily function and work productivity. Some of these symptoms, like fatigue and pain, have not been well studied in Cushing’s patients, and need more attention.” Geer and colleagues administered a cross-sectional survey to 55 adults aged 21 years and older who had been diagnosed with Cushing’s syndrome at least 6 months before the survey and were receiving at least one pharmacologic therapy for their disease (85% women; mean age, 43.4 years). The survey was conducted online from June to August 2021. Five patient-reported outcome scales were included. The CushingQoL was used to analyze quality of life, a visual analog scale was included to assess pain, the Brief Fatigue Inventory was used to measure fatigue, the Sleep Disturbance v1.0 scale assessed perceptions of sleep and the PROMIS Short Form Anxiety v1.0-8a scale was used to measure fear, anxious misery, hyperarousal and somatic symptoms related to arousal. Participants self-reported the impact of Cushing’s syndrome on daily life and their physician’s level of awareness of Cushing’s syndrome. Some symptoms decline in severity over time Of the study group, 81% had pituitary or adrenal tumors, and 20% had ectopic adrenocorticotropic hormone-producing tumors; 80% of participants underwent surgery to treat their Cushing’s syndrome. The frequency of reported symptoms did not change from Cushing’s syndrome diagnosis to the time of the survey. The most frequently reported symptoms were weight gain, muscle fatigue and weakness and anxiety. Participants reported a decline in symptom severity for weight gain, muscle fatigue and weakness and menstrual changes from diagnosis to the survey. Though symptom severity declined, none of the three symptoms were entirely eliminated. Adults did not report declines in severity for other symptoms. Hirsutism and anxiety were reported by few participants, but were consistently scored high in severity among those who reported it. There were no changes in patient satisfaction with medications from their first appointment to the time of the survey. “It was surprising that anxiety and pain did not improve with treatment,” Geer said. “A quarter of patients at baseline reported anxiety and this percentage was exactly the same after treatment. Same for pain — nearly a quarter of patients reported pain despite treatment. While the presence of anxiety has been well-documented in Cushing’s patients, pain has not, and needs further study.” Nearly half of primary care providers unable to diagnose Cushing’s syndrome All participants reported having at least one challenge with being diagnosed with Cushing’s syndrome. Of the respondents, 49% said their primary care provider was unable to diagnose their Cushing’s syndrome and 33% initially received the wrong diagnosis. Physicians referred 49% of participants to a specialist, and 39% of adults said their doctor lacked knowledge or understanding of their condition. The study group had a moderate level of quality of life impairment as assessed through the CushingQoL scale. The mean pain score was 3.6 of a possible 10, indicating low levels of pain. Moderate to severe levels of fatigue were reported by 69% of participants. Self-reported sleep and anxiety scores were similar to what is observed in the general population. Participants said sexual activity, self-confidence and life satisfaction were most impacted by a Cushing’s syndrome diagnosis. Adults experienced symptoms a mean 16 days in a typical month and saw their outpatient physician an average of six times per year. Those who were employed said they miss 2 days of work per month, or about 25 days per year, due to Cushing’s syndrome. “Longitudinal assessment of clinically relevant patient-reported outcomes based on validated measures and coupled with biochemical and treatment data is needed in a large cohort of Cushing’s patients,” Geer said. “This will allow us to identify clinically meaningful changes in symptom burden within each patient, as well as predictors of outcomes — which patients improve on which symptoms, and which patients do not feel better despite biochemical normalization. We need to improve our ability to help our patients feel better, not just achieve normal cortisol levels.” For more information: Eliza B. Geer, MD, can be reached at geere@mskcc.org. From https://www.healio.com/news/endocrinology/20230830/adults-with-cushings-syndrome-report-high-burden-of-illness-despite-ongoing-treatment

Introduction: The differential diagnosis between Cushing’s disease (CD) and ectopic ACTH syndrome (EAS) is complex, and bilateral inferior petrosal sinus sampling (BIPSS) is considered the gold-standard test. However, BIPSS with corticotropin-releasing hormone (CRH) stimulation is rarely available. Objective: This retrospective cohort study aimed to assess the accuracy of the inferior petrosal sinus to peripheral ACTH gradient (IPS:P) before and after desmopressin stimulation for the differential diagnosis of ACTH-dependent Cushing’s syndrome (CS), applying different cutoff values. Methods: A total of 50 patients (48 with CD and 2 with EAS) who underwent BIPSS were included in this study. The sensitivity and specificity of IPS:P in BIPSS before and after desmopressin stimulation were evaluated. Various cutoff values for IPS:P were examined to determine their diagnostic accuracy. Results: Using the traditional IPS:P cutoff, the sensitivity was 85.1% before stimulation, 89.6% after stimulation, and a combined sensitivity of 91.7%. Applying cutoff values of IPS:P >1.4 before and >2.8 after stimulation, the sensitivity was 87.2% and 89.6%, respectively, with a combined sensitivity of 91.7%. Receiver operating characteristic (ROC) curve analysis determined optimal cutoff values of 1.2 before stimulation and 1.57 after stimulation, resulting in a sensitivity of 93.6% and 93.8%, respectively, with a combined sensitivity of 97.9%. Specificity remained at 100% throughout all analyses. Among the 43 patients who responded positively to stimulation, 42 (97.7%) did so within the first three minutes, and all 43 (100%) did so within the first five minutes. None of the assessed clinical variables predicted the ACTH response to stimulation in BIPSS with statistical significance. Discussion: ACTH stimulation with desmopressin during BIPSS improves the accuracy of IPS:P, making it a valuable tool for investigating ACTH-dependent Cushing’s syndrome. Considering the low risk of complications, we recommend the use of desmopressin stimulation during BIPSS for the differential diagnosis of ACTH-dependent CS. Introduction Cushing Syndrome (CS) is a rare disease that results from chronic exposure to elevated cortisol levels. It can be caused by either endogenous or exogenous factors, and its incidence is estimated to be 0.7-3.2 cases per million per year (1, 2). The mortality rate for CS is elevated and may remain higher than the general population even after remission of hypercortisolism (3, 4). The causes of endogenous CS are traditionally classified into two categories: ACTH-dependent (about 80-85% of cases) and ACTH-independent (15-20% of cases) (5). The most common cause of ACTH-dependent CS (75-80% of cases) is Cushing Disease (CD), which is characterized by a corticotropic pituitary adenoma. The remaining cases (15-20%) of ACTH-dependent CS are caused by ectopic ACTH syndrome (EAS), which occurs when tumors of various sites, histological differentiation, and aggressiveness produce ACTH. There are also exceptionally rare cases (<1%) of ectopic CRH-producing tumors (5, 6). CS diagnosis is a complex and challenging pathway due to the variable pattern of hormonal findings, the non-specificity of clinical presentation, particularly in mild hypercortisolism states (7), and the technical limitations of diagnostic tests. Once CS is confirmed, it should be differentiated between ACTH-dependent or -independent cases (8). ACTH levels <10 pg/ml suggest an adrenal cause; ACTH levels >20 pg/ml suggest ACTH-dependent causes; and levels between 10-20 pg/ml are considered indeterminate, requiring additional tests to establish the etiology (5, 8). When ACTH-dependency is confirmed, the next diagnostic step is the differentiation between CD and EAS. In this step, non-invasive tests are initially recommended, such as the CRH test (CRH-t), the 8 mg dexamethasone suppression test (DST-8 mg), and a pituitary magnetic resonance imaging (MRI) (5, 8). These tests, however, presents heterogenous results, depend on the availability of CRH, restricted in many countries including Brazil, and present low discriminatory power (9, 10). An alternative to CRH-t is the use of desmopressin, which stimulates ACTH release in most patients harboring ACTH-secreting pituitary adenomas. The use of this stimulus for the differential diagnosis of CD vs EAS is controversial, since studies have demonstrated that EAS patients may present ACTH elevation following desmopressin administration (11–14). The DST-8 mg is widely available; however it also presents limitation due to the variability of criteria used; furthermore, it has shown insufficient discriminatory capacity in some studies (15, 16). Pituitary MRI fails to detect adenomas in CD patients in about 30-50% of cases even with modern technology equipment (17); moreover, it may also generate false-positive results since pituitary incidentalomas are common in the population, including macroadenomas (18). In cases of conflicting non-invasive test results and unavailability of other methods, bilateral inferior petrosal sinus sampling (BIPSS) should be performed to detect a central-to-peripheral ACTH gradient that allows the localization of the ACTH production (5). Some authors and guidelines recommend performing BIPSS in all patients with pituitary lesions < 6 mm demonstrated on MRI (5, 8, 19), whereas others suggest BIPSS should routinely be performed, especially to guide surgical therapy of CD (20–23). Thus, the procedure is considered the gold-standard in the differential diagnosis of ACTH-dependent CS, preferentially performed with CRH or, less frequently, with desmopressin. The use of CRH is a limiting factor since it is unavailable in many countries. On the other hand, although used in some medical centers, desmopressin as a stimulus for BIPSS is still poorly debated and assessed in the literature, and its utility in this setting remains uncertain since studies validating it in different populations and in larger series are still lacking (8, 24–26). A recent study evaluating desmopressin in a large cohort of patients proposed new diagnostic criteria, questioning the need of stimulus with the new cut-offs (27). Thus, the aim of this study is to assess the role of central-to-peripheral ACTH gradient after stimulus with desmopressin during BIPSS for the differential diagnosis of ACTH-dependent CS in a cohort of patients followed-up in a referral center for CS in Brazil. Patients and methods Patients Between 1998 and 2020, 107 patients with ACTH-dependent CS were retrospectively evaluated at the Neuroendocrinology clinic of a tertiary center in Southern Brazil for BIPSS under desmopressin stimulation during initial diagnostic evaluation or after recurrence. Of these, 58 patients underwent BIPSS with desmopressin, 50 of which for the initial diagnostic evaluation, 7 after recurrence and 1 after emergency adrenalectomy. Eight patients who underwent BIPSS were excluded for insufficient data regarding final etiologic diagnosis (lack of histopatological confirmation, lack of biochemical remission 6 months after surgery, or lack of remission after radiotherapy). Finally, 50 patients were included in the analysis. The present study was conducted in compliance with the principles laid down in the Declaration of Helsinki and was approved by the Hospital de Clínicas de Porto Alegre Ethics Committee. Diagnosis of CS and ACTH-dependency status After exhaustive screening for exogenous glucocorticoid administration, CS diagnosis was based on the presence of at least two of the following conditions: cortisol after low-dose dexamethasone suppression test (either 1 mg overnight or 0.5 mg 6/6 hours for 48h) > 1.8 µg/dL (DST-1mg); 24-h urinary free cortisol (UFC) or late night salivary cortisol consistently elevated in at least two samples (8). Additionally, late night serum cortisol > 7.5 µg/dL (8) and a desmopressin test (DES-t) with a peak ACTH > 71.8 pg/mL or an increase in ACTH ≥ 37 pg/mL from baseline (28) were also considered suggestive of CS. After clinical and biochemical diagnostic confirmation of CS, plasma ACTH measurement classified CS into ACTH-dependent (ACTH > 20 pg/dL) or ACTH-independent (ACTH < 10 pg/dL). Values between 10-20 pg/dL were considered indeterminate and new samples were obtained for correct classification (8). Next, patients diagnosed with ACTH-dependent CS underwent pituitary MRI for the identification of an adenoma. Due to the unavailability of CRH-t, it was rarely performed. The DES-t for the differential diagnosis of CD and EAS was considered predictive of CD when the increase was > 20% in cortisol or >35% in ACTH after stimulus. In virtue of its low accuracy, DST-8 mg was only performed in a few cases. Patients with inconclusive or negative imaging, those with adenomas < 6 mm or those with adenomas > 6 mm but discordant non-invasive tests were submitted to BIPSS with sampling of ACTH at baseline and after desmopressin stimulus. After investigation, patients with a suggestive diagnosis of CD underwent transsphenoidal surgery. Histological confirmation of a pituitary adenoma staining positive for ACTH was considered the gold-standard for diagnosis. Additionally, patients with inconclusive or absent histological specimen who exhibited clinical and biochemical remission 6 months after surgery or who remitted after pituitary radiotherapy were also considered diagnosed for CD. The EAS cases were confirmed based on surgical excision or biopsy of tumoral lesions confirming the presence of ACTH-staining neoplastic cells. Bilateral inferior petrosal sinus sampling The procedure was performed in the presence of documented hypercortisolism, in an angiography room, under sedation with fentanyl and midazolam, and by a qualified professional in interventional radiology. Initially, bilateral common femoral venipuncture was performed, maintained with 6 French (F) introducers. Then, ascending catheterization of the superior vena cava and internal jugular veins was performed with a 5F vertebral catheter and hydrophilic guidewire, with final positioning of the catheter tip at the level of the inferior petrosal sinuses. Angiographic confirmation was performed after injection of 10 ml of diluted nonionic contrast under digital subtraction, demonstrating bilateral sinus and sellar region opacification. In situations of fine-caliber inferior petrosal sinuses, a coaxial microcatheter was used for a better distal reach of the required topography. Heparinization was not usually necessary in this technique, only sequential washing of the catheters was performed between the sampling times with saline solution with 2 ml of heparin for each 1000 ml of solution. Samples were collected after washing the catheters at baseline. Then, 10 µg of desmopressin was administered intravenously and samples were collected after one, three, five, and 15 minutes. In some cases, the sampling times were slightly different, but always with one sampling at baseline and at least 3 samplings after stimulation. All samples were collected in ice-cold tubes, kept on ice and then centrifuged in a refrigerated centrifuge and frozen at -8°C until ACTH measurement, which occurred immediately after the end of the procedure. After the samplings, the catheters and introducers were removed, followed by manual compression of the inguinal region at the puncture site for 10 minutes, until complete hemostasis. After compression, a compressive dressing was placed at the puncture site and the patients remained at bed rest without flexing the thigh for 6 h. Our routine protocol in performing the BIPSS did not include the concomitant measurement of prolactin as suggested in some previous studies in the literature. Hormone assays Until April 2004, cortisol was measured using a commercially available radioimmunoassay (RIA) kit (Diagnostic Systems Laboratories, Webster, TX, USA). From May 2004 to March 2010, the method was modified to an electrochemiluminescence immunoassay (ECLIA) kit (Modular Analytics E 170; Roche, Mannheim, Germany). From March 2010 to February 2014, cortisol was measured by chemiluminescence immunoassay (ADVIA Centaur XP Immunoassay System, Tarrytown, NY, USA). From February 2014 to October 2019, the method was Competitive Electrochemiluminescence. (Roche e602 equipment line). From October 2019 until the end of the study, the method was Microparticle Chemiluminescent Immunoassay. (Abbott equipment line). ACTH measurements up to February 2000 were performed by commercially available RIA. From February 2000 to April 2015, the method was chemiluminescence with the Immulite 1000 equipment. From May 2015 to April 2018, the method was electrochemiluminescence with the Roche e602 equipment. From May 2018 to August 2019, the method was sandwich electrochemiluminescence using the Roche e602 equipment. From August 2019 until the end of the study, the method was chemiluminescent immunoassay in the Immulite 2000 equipment. These assay differences do not show a large variation from normal values and as samples collected from the same patient were always analyzed with the same assay, the calculations of different indexes of central versus peripheral samplings did not change as a result of the trials. Of the cases studied, ACTH was measured by RIA in 1 patient, by Immulite 1000 in 35 patients, by Roche e602 via electrochemiluminescence in 9 patients, by Roche e602 via sandwich electrochemiluminescence in 4 patients and by Immulite 2000 in 1 patient. The basal ACTH and UFC values, therefore, are presented according to the percentage above the ULN according to each methodology used at each moment. For the calculation of the ACTH inferior petrosal sinus to peripheral gradient (IPS:P), however, absolute values were used since the ratios are calculated for the same patient using the same assay. Statistical analysis The Kolmogorov-Smirnov test was used to assess the distribution of variables. Continuous variables with normal distribution are presented as mean ± standard deviation (SD). Continuous variables with asymmetric distribution are shown as median and interquartile range (IQR). Categorical variables were compared using Fischer’s exact test. The comparison of continuous variables was performed using the Mann-Whitney test. ROC curves were used to assess the ability of the IPS:P gradient to discriminate between CD and EAS, and the Youden index was used to define optimal cutoffs. Sensitivity and specificity were calculated for the different criteria analyzed. Statistical analyzes were performed using the SPSS 24.0 program (statistical package software, SPSS Incorporation, Chicago, IL, USA). Differences were considered significant when p<0.05. Results Patient characteristics are shown in Table 1. During the study period, 50 patients with a confirmed diagnosis of ACTH-dependent CS whose etiology could be confirmed through histopathological or biochemical data (remission after 6 months of surgery or after radiotherapy) who had undergone the BIPSS were included. The mean age (SD) at diagnosis was 38.22 (15.56) years, 39 patients (78%) were female, and 48 patients had CD and 2 EAS. Table 1 Table 1 Characteristics of studied patients. In the imaging results, 23 (46%) were microadenomas, among which 15 were < 0.6cm (65.2% of microadenomas), 8 were macroadenomas (16%), and 19 had negative or inconclusive imaging (38%). One of the patients with EAS had an image suggestive of a 0.4 cm microadenoma on MRI. Regarding macroadenomas, the indication for BIPSS was proposed based on the following situations: 3 presented with a clinical picture of EAS, including 2 with systemic lesions suspicious for neoplasia, 3 presented imaging characteristics that were somewhat atypical for adenomas, 1 was associated with a brainstem vascular lesion and one was a recurrent disease with postsurgical alteration and residual lesion. BIPSS was performed in 44 patients who had not yet undergone investigation or treatment and in 6 patients who had been previously treated for CD but had relapsed during follow-up. No complications were recorded in any of the cases submitted to BIPSS. There were no thromboembolism events related to the procedure. At baseline (before stimulation), 49 patients were evaluated (1 patient with CD had samples collected, but his results were not properly recorded). The median IPS:P gradient at baseline was 6.62 (IQR 2.46-11.36) in patients with CD and 1.14 (IQR 1.10-1.14) in patients with EAS (p=0.01). Using the IPS:P>2 gradient criteria, 40 of 47 patients with CD were positive and none of the 2 patients with EAS were positive, resulting in 85.1% sensitivity (95% confidence interval (CI) 71.1-93.3%) and 100% specificity. After stimulation with desmopressin, all 50 patients were evaluated. The median SPI:P gradient after stimulation was 29.46 (IQR 15.39-61.50) in patients with CD and 1.26 (min-max 1.25-1.28) in patients with EAS (p=0.01). In patients with EAS, the highest ACTH peak was 537 pg/mL (109.5% increase from baseline), while in patients with CD, the lowest increase from baseline was 19.48%. Using the IPS:P gradient criteria > 3, 43 of 48 patients with CD were positive, and none of the 2 patients with EAS were positive, resulting in 89.6% sensitivity (95%CI 76.5-96.1%) and 100% specificity. When evaluating patients who were positive at baseline and/or after stimulation in a combined manner, 44 of 48 CD patients were positive, whereas no EAS patients were positive. The overall sensitivity, therefore, was 91.7% (95%CI 79.1-97.3%), and the specificity was 100%. Of the 9 negative patients at baseline, 3 (33.33%) became positive after stimulation. Among the 43 patients who tested positive after the stimulus, 42 (97.7%) had already tested positive up to the third minute, and 100% of the patients were positive up to the fifth minute (Figure 1), totaling 86% of the total sample. Of the 3 patients whose stimulation was necessary, 2 had microadenomas and 1 had macroadenomas. In the two patients with EAS, the time of peak of ACTH was at 1 minute for patient 1 (31.1% increase from baseline) and at 3 minutes for patient 2 (109.5% increase from baseline). Figure 1 Figure 1 Time (minutes) until obtaining IPS:P gradient values of ACTH considered positive response of BIPSS after stimulation with desmopressin. When assessing only the 23 patients with microadenoma, 20 of 22 patients with CD were positive at baseline, and the patient with EAS and 0.4 cm microadenoma was negative, resulting in 90.9% sensitivity (95%CI 69.37-98.4%), while maintaining 100% specificity. After stimulation, all 22 patients with CD were positive and the only patient with EAS and microadenoma was negative, resulting in 100% sensitivity (95%CI 81.5-100%) while maintaining 100% specificity. When only microadenomas < 0.6 cm were evaluated, 12 of 14 CD patients were positive at baseline, and the patient with EAS and 0.4 cm microadenoma was negative, resulting in 85.7% sensitivity (95%CI 56.2-97.5), with 100% specificity. After stimulation, all 14 patients with CD were positive, and the patient with EAS and microadenoma was negative, resulting in a sensitivity of 100% (95%CI 73.2-100%) while maintaining 100% specificity. All eight patients with microadenomas >0.6cm were already positive at baseline and remained positive after stimulation (100% sensitivity and 100% specificity). Thus, only patients with microadenoma <0.6 cm improved sensitivity after stimulation. Among the 8 patients with macroadenoma, sensitivity was 75% at baseline and remained the same after stimulation. However, when assessed for need for stimulation, only one patient with macroadenoma benefited, but sensitivity did not increase because a patient who was positive at baseline became negative after stimulation. Assessing all patients with positive imaging on MRI (micro or macroadenomas, n = 31), 26 of 30 CD patients were positive at baseline, and the patient with EAS and microadenoma was negative, resulting in 86.7% sensitivity and 100% specificity. After stimulation, 28 of 30 CD patients were positive and the patient with EAS and microadenoma remained negative, resulting in 93.3% sensitivity and maintaining 100% specificity. The combined sensitivity (baseline or after stimulus) in this group of patients was 96.7%. Among the 19 patients with negative imaging, 18 had baseline results and were evaluated. Baseline sensitivity was 82.4%. After stimulation, data from 19 patients were evaluated and resulted in a sensitivity of 83.3%. When the patients with negative imaging (n=19) and those with microadenomas <0.6 cm (n=15) were analyzed together, which represent the most difficult cases in clinical practice, we observed that the IPS:P gradient >2 at baseline resulted in sensitivity of 83.9% and 100% specificity. After stimulation, the IPS:P >3 gradient had a sensitivity of 90.6% while maintaining 100% specificity. After assessing the traditionally proposed criteria, the analysis was performed using the criteria proposed by Chen et al. (27). Using the IPS:P gradient at baseline > 1.4, 41 of 47 CD patients were positive and none of the EAS patients were positive, resulting in 87.2% sensitivity (95%CI 73.5-94.7%) while maintaining 100% specificity. After stimulation, using the IPS:P>2.8 gradient criteria, 43 of 48 patients with CD were positive, resulting in 89.6% sensitivity (95%CI 76.5-96.1%), strictly the same as the traditional criteria maintaining 100% specificity. When evaluating patients who were positive at baseline and/or after stimulation, 44 of 48 patients with CD were positive, and no patient with EAS was positive, resulting in 91.7% overall sensitivity (95%CI 79.1-97.3%), the same as the traditional criteria. Finally, only 2 of 49 patients who were negative at baseline became positive after stimulation. To establish institution-specific cut-off points, a ROC curve was performed to assess the accuracy of the central/peripheral ACTH gradient in BIPSS in our cohort of patients. For the IPS:P gradient at baseline, the cut-off point with the highest accuracy was 1.2, whereas for the IPS:P gradient after stimulation, the cut-off point with the highest accuracy was 1.57 (Figure 2). Using these cut-off points, 44 of 47 CD patients were positive at baseline and no EAS patients were positive, resulting in 93.6% sensitivity (95%CI 81.4-98.3%), while maintaining 100% specificity. After stimulation, 45 of 48 CD patients were positive and no EAS patients were positive, resulting in 93.8% sensitivity (95%CI 81.8-98.4%), with 100% specificity (Figure 3). When evaluating patients who were positive at baseline and/or after stimulation, 47 of 48 CD patients were positive and no EAS patients were positive, resulting in an overall sensitivity of 97.9% (95%CI 87.5-99.9%) With 100% specificity. Finally, only 2 patients who were negative at baseline became positive after stimulation. Figure 2 Figure 2 ROC curve of baseline IPS:P values in BIPSS in the investigation of ACTH-dependent CS. Figure 3 Figure 3 ROC curve of IPS:P values after stimulation with desmopressin in BIPSS in the investigation of ACTH-dependent CS. In the comparison between the traditional criterion and our study criterion, the baseline sensitivity changed from 85.1 to 93.6%. After stimulation, baseline sensitivity changed from 89.6 to 93.8%, respectively. A summary of the sensitivity results with the different diagnostic criteria is presented in Table 2. Table 2 Table 2 Sensitivity of BIPSS with traditional criteria and with present study criteria. Technical difficulties or anatomical variations were found in 6 patients undergoing BIPSS. Among the 43 cases with a positive IPS:P gradient, 3 had anatomical variations and 1 had some technical difficulty. Of the 5 cases in which the IPS:P gradient did not occur (false-negatives), 1 presented anatomical variation and 1 presented some technical difficulty during the test. Among the 6 patients who underwent BIPSS after recurrence, all had a final diagnosis of CD, and only 1 was negative on BIPSS. Of the 50 patients evaluated, 43 had undergone DES-t as part of the diagnostic workup, of which 41 were later diagnosed with CD and 2 with EAS. Forty patients were considered responsive in DES-t, 38 patients with CD and 2 patients with EAS. Among the 40 responsive patients, 34 (85%) were also positive in BIPSS, all with a final diagnosis of CD. The 3 non-responsive patients in DES-t presented a positive response in BIPSS after desmopressin. Of the 6 patients who were positive in DES-t but negative in BIPSS, 2 were patients with EAS. Of the 4 patients with CD, 2 had normal petrosal sinus anatomy, 1 had a report of some anatomical variation, and 1 had a report of technical difficulties during BIPSS. Thus, DES-t was not able to predict response to desmopressin during BIPSS (p>0.9999). When comparing the ACTH values at baseline, 3, 5 and 10 minutes after stimulation in BIPSS, there was no significant difference between the group with positive versus negative DES-t, as well as no difference in the time to positivity between the groups, adenoma size, and number of patients with negative imaging. In addition, the clinical variables evaluated (ACTH, UFC, DST-1mg, baseline cortisol, adenoma size) were not able to significantly predict response to stimulus. Discussion In this study, the use of BIPSS with ACTH measurements at baseline and after stimulation with desmopressin in the differential diagnosis of the ACTH-producing source in a sample of 50 patients with ACTH-dependent CS and inconclusive non-invasive tests resulted in 85.1% baseline sensitivity, increasing to 89.6% after stimulation, maintaining 100% specificity when applying traditional IPS:P≥2 criteria at baseline and ≥3 after stimulation (29). When combined, the baseline and/or stimulated sensitivity results were 91.7%. Results of meta-analyses that combined studies performed with CRH stimulation and desmopressin indicate that the sensitivity of BIPSS ranges from 86-97% and the specificity from 89-100% (27, 30). Published studies with desmopressin are generally small, with a variable number of cases of EAS, different indications for BIPSS, and variable diagnostic criteria. In a study with a sample of 56 patients with ACTH-dependent CS and negative imaging, using the criterion of IPS:P≥2 at baseline and IPS:P≥3 after stimulation with desmopressin, the combined sensitivity was 92.1% and 100% specificity, similar to the findings of the present study (25). Smaller studies that also used desmopressin stimulation found similar (26, 31–33) or slightly higher sensitivities (34, 35). Studies performed exclusively in pediatric patients were less uniform, with one of them reporting similar results to studies that included adults (36) and another study demonstrating lower sensitivity in adult population (37). Our institution’s optimal cut-off points, determined by analyzing the ROC curve, were IPS:P≥1.2 at baseline and ≥1.57 after stimulation. This resulted in 93.6% baseline sensitivity (it was 85.1% with IPS:P≥2), and 93.8% after stimulation (was 89.6% with IPS:P≥3), and a combined sensitivity of 97.9% (it was 91.7%), maintaining specificity at 100%. Despite the increased sensitivity, these criteria should be used with caution, since the number of cases with EAS was small. The IPS:P gradient at baseline and after stimulation achieved in patients with EAS in some studies with desmopressin would exceed the cutoffs found by us (24, 25, 27), which would incorrectly classify these patients as CD. Before adopting the new values in our institution, therefore, more patients with EAS are necessary to validate these criteria. Also using the ROC curve, Castinetti et al. evaluated 43 patients with ACTH-dependent SC (36 DC and 7 EAS) and established the criteria of IPS:P>2 at baseline or after stimulation, obtaining a sensitivity of 86% at baseline and 97% after stimulation with desmopressin, not mentioning the combined sensitivity. The study, however, showed 85% specificity at baseline, given that a patient with EAS had a 3.33 gradient (24). In addition to applying the traditional criteria, Machado et al. also used ROC curve analysis to establish cut-off points, finding an IPS:P≥1.45 at baseline (88.2% sensitivity) and ≥ 2.04 after stimulation (92.2% sensitivity) as optimal, both with 100% specificity, although the authors did not recommend the use of these new values (25). The results of these studies using the ROC curve suggest that lower cutoff points, both at baseline and after stimulation, can improve sensitivity without compromising specificity. However, a study that performed a ROC curve in patients stimulated with CRH found an optimal 2.10 baseline cut-off, slightly higher than the traditional one of 2, although the post-stimulation cut-off point was 2.15, lower than the one usually used (38). A study with desmopressin, in turn, found values in the ROC curve of 1.76 at baseline, lower than the traditional one, but ≥3.9 after stimulation, higher than the gradient of three usually used, increasing baseline sensitivity but keeping the sensitivity after stimulation unchanged (32). The largest published study evaluating BIPSS with desmopressin stimulation evaluated 226 patients with CD and 24 with EAS (27). Applying the IPS:P>2 criteria at baseline and >3 after stimulation, the sensitivity was 87.2 and 94.2%, respectively, while maintaining 100% specificity. The combined sensitivity was 96.5%. In this series, 3 cases of EAS reached gradients greater than 2 after stimulation, which suggests that cut-off points equal to or lower than this may decrease specificity. The authors also performed an ROC curve, determining the cutoff point of >1.4 at baseline and >2.8 after stimulation. In this analysis, the sensitivity at baseline was 94.7% and 96% after stimulation, resulting in a combined 97.8% sensitivity, higher than that found with the traditional criteria. According to the authors, with these cut-off points, only 7 patients benefited from the stimulus. After this publication, no other studies have tested these new cutoffs. Our study was the first, therefore, to assess the new values. In our series, using the cutoff point of >1.4 at baseline and >2.8 after stimulation, the sensitivity was 87.2 and 89.3%, respectively, and the combined sensitivity was 91.7%, thus slightly improving the sensitivity at baseline with little change after stimulation. In an attempt to identify predictors of need for stimulation, Chen et al. found that patients requiring stimulation had adenomas < 0.6 cm or negative imaging. In addition, patients who required stimulation had lower IPS ACTH levels and did not lateralize. These data, however, are obtained only after performing the BIPSS, which makes their use in practice unfeasible (27). In our series, among patients with microadenomas, only those with lesions <0.6 cm benefited from the stimulus. Patients with negative imaging had a small increase in sensitivity. A patient with a macroadenoma also benefited from the stimulus, although the sensitivity of the cases with macroadenoma did not change, as a positive patient at baseline became negative after the stimulus. Despite current recommendations suggesting to perform BIPSS in patients with adenomas < 0.6 cm or with negative/inconclusive imaging results (8, 39), Chen et al. identified 2 patients with EAS and adenomas > 0.6 cm who would be misdiagnosed with CD if the 0.6 cm threshold were respected. Therefore, they suggest performing BIPSS in all patients with ACTH-dependent CS (27). Given the relevance of EAS cases in this study, a discussion about the current size criteria for indicating BIPSS should be undertaken. Of our 50 patients, 43 (41 CD and 2 EAS) underwent DES-t prior to BIPSS, and 40 were considered responsive, including the two cases of EAS. Among the responders, 34 patients also responded to the stimulus during the BIPSS, all of them with CD. The 3 patients who did not respond to the peripheral stimulus were, however, positive in the BIPSS. The lack of correlation between the DES-t results and the BIPSS may be related to the different sampling intervals in the two exams (short intervals in the BIPSS and long intervals in the peripheral test). Considering that the majority (86%) of our patients performed both tests, it is possible to conclude that the DES-t did not help in the prediction of response to the central stimulus, which makes the use of peripheral test results debatable for this purpose. Of the BIPSS studies with desmopressin, only one described the results of DES-t, although it did not perform any specific analysis of the relationship with BIPSS (36). The study differs from ours, also, as it only evaluated pediatric patients. Although BIPSS is still considered the gold standard in the differential diagnosis of ACTH-dependent CS, some authors have suggested that the procedure should be indicated only in cases in which t-CRH was negative (40, 41). Recent studies have evaluated non-invasive strategies combining t-CRH, DES-t, TSD-8mg, and imaging to reduce the need for BIPSS. Strategies that resulted in a positive predictive value of 100%, however, included t-CRH as part of the diagnostic process (42, 43), which makes adherence to this diagnostic modality inapplicable in many countries due to the unavailability of CRH. In one of these studies, the combination of TSD-8mg with DES-t, which would be possible in Brazil, was inferior to the combination of DES-t with t-CRH or t-CRH with TSD-8mg (43). The low number of patients undergoing TSD-8mg in our study did not allow the evaluation of this strategy. Although not recommended as a test in the differential diagnosis of the etiology of ACTH-dependent SC, DES-t seems promising as a marker of long-term postoperative outcome and as an early marker of recurrence (44), which encourages further studies in these circumstances. Despite there have been reports of thromboembolic events related to BIPSS that occurred heparin (45, 46), it is a very rare complication. The administration of desmopressin, which increases coagulation factor VIII and von Willebrand factor (47), has raised concerns about the potential for increased incidence of thromboembolic events during BIPSS. This is due to the fact that desmopressin is associated with the hypercoagulable state of CS (48) and may also interfere with VIII and von Willebrand factors. The study by Chen et al, the largest published with desmopressin to date, did not record any case of thromboembolism, even without routine anticoagulation during the procedure (27). In our study, performed without routine anticoagulation, there were also no thromboembolic events. The only desmopressin BIPSS study that recorded thromboembolic events routinely used heparin during the procedure (25). Thromboembolic events, therefore, do not appear to be an additional concern when using desmopressin, with or without the use of heparin during the procedure. The decision regarding the use or not of anticoagulants during BIPSS should be a decision of each institution and based on the usual anticoagulation recommendations. In our study, we did not perform the concomitant dosage of prolactin in samples collected from the inferior petrosal sinuses, a procedure that potentially reduces false negatives, as advised by some authors based on studies with CRH (49–51) and a study with desmopressin (31). These findings, however, were not confirmed by all groups, both with CRH (52) and with desmopressin (32), and their applicability depends on further studies to define its role. In our study, a total of 3 patients who were negative at baseline benefited from the stimulus, As they became positive, 2 of them with microadenomas and one with macroadenoma. The study by Chen et al. questions the use of routine stimulation in all patients to reduce the risks and the duration of the procedure, potentially reducing complications. The authors argue that, when using the IPS:P>1.4 criterion at baseline, the sensitivity was high enough to classify most patients, with the exception of 7 patients with adenoma <0.6 cm who needed stimulation (27). Our study, however, would have misclassified a case with CD and macroadenoma as EAS if the stimulus had not been performed. The assessment of the need for stimulation in cases of CS with macroadenoma is limited since most studies performed the BIPSS only in patients with lesions < 0.6 cm or negative imaging, preventing a more comprehensive assessment. Considering that BIPSS is currently still the gold standard in the differential diagnosis of ACTH-dependent CS, even small gains in sensitivity should be considered important since incorrect classification of patients can lead to inappropriate treatments and potentially fatal delays in the resolution of hypercortisolism. Considering that BIPSS is generally well tolerated and the rate of serious complications is low (53), other strategies to reduce the risks of the procedure that do not involve avoiding the stimulus seem necessary. In this context, it is important to evaluate the time interval between the infusion of the secretagogue and the positive test result. In our study, 97.7% of the patients who tested positive after stimulation were already positive in the third minute and 100% of the patients were positive until the fifth minute, demonstrating that there seems to be no benefit in prolonging the test beyond this period. All of the few studies on BIPSS with desmopressin have directly or indirectly reported a similar time to positivity and for peak ACTH (i.e., positive up to 3-5 minutes) (26, 33, 35, 37). Stimulating patients for a maximum time of 5 minutes considerably reduces the procedure time without neglecting the sensitivity gain resulting from the stimulation and may, therefore, be a strategy to potentially reduce the risk of complications. Our study evaluated a sample of patients whose BIPSS indication was more comprehensive since the unavailability of t-CRH and the low accuracy of TSD-8mg limited the use of non-invasive tests. The wide heterogeneity existing in the BIPSS studies regarding the characteristics of the evaluated patients (primary diagnosis or recurrence), the BIPSS technique (sampling times, anticoagulant use, material used, laboratory assays, cut-off points, type of secretagogue) makes direct comparisons difficult. Conducting multicenter prospective studies with a greater sample of EAS patients is necessary to improve our understanding of the best cut-off points and procedure duration. The present study has some limitations, as expected in the complexity of CS investigation. Our main limitation is that the low prevalence of EAS that underwent BIPSS, resulting from the rarity of this condition, may explain the high specificity when applying the cutoff points indicated by the ROC curve, and the application of these new gradients of IPS:P depends on validation in larger samples of EAS. Lower specificity may result from poor responsiveness to the secretagogue (desmopressin or CRH), cyclic CS during periods of normal cortisol secretion or due to anomalous venous drainage (54). Retrospective data collection and analysis prevented access to complete information for all patients. There were differences over time in terms of sampling times, although at least 3 different samplings were always performed throughout the study period. We highlight that, in this study, we did not discuss the data regarding the eventual lateralization of the basal ACTH values and after stimulation with desmopressin to guide the location of the pituitary adenoma in the transsphenoidal surgery. This utility of the BIPSS has been less and less recommended in the literature due to the imprecision of the results, especially due to the existence of venous communications between the cavernous sinuses and the instability and intensity of blood aspiration for sample collection. In conclusion, in BIPSS with ACTH dosage, the use of stimulation with desmopressin increases the sensitivity of the test from 85.1% to 89.6%, reaching 100% in the sub-analysis of microadenomas. In spite of being small, this increase is useful in the investigation of ACTH-dependent CS, a clinical situation in which gains in diagnostic sensitivity are very important. Additionally, considering the low risk of complications and the possibility to interrupt the test within 5 minutes, as demonstrated in our study, our data recommend the use of stimulation with desmopressin in the BIPSS in the differential diagnosis of ACTH-dependent CS. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Ethics statement The studies involving human participants were reviewed and approved by Hospital de Clínicas de Porto Alegre Ethics Committee. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Author contributions TA, TR and MC conceived the study and designed the research. TA conducted the data collection and database management. TA performed the data analysis. LS, MF and FG performed the BIPSS procedures. TA, TR, FC and MC contributed to the interpretation of the results. TA and MC drafted the manuscript. FC critically revised the manuscript. All authors read and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version. Funding This work was supported by the Research Incentive Fund (FIPE) of Hospital de Clínicas de Porto Alegre and the Postgraduate Program in Medical Sciences: Endocrinology (PPG ENDO) from Universidade Federal do Rio Grande do Sul. Acknowledgments The authors would like to acknowledge the contributions of Guilherme Alcides Flores Soares Rollin, Arthur Boschi, and Camila Viecceli to the data collection process. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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Second-line tests in the diagnosis of adrenocorticotropic hormone-dependent hypercortisolism. Ann Lab Med (2021) 41(6):521–31. doi: 10.3343/alm.2021.41.6.521 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: Cushing’s syndrome, Cushing’s disease, ectopic ACTH syndrome, bilateral inferior petrosal sinus sampling, ACTH, desmopressin Citation: Almeida TSd, Rodrigues TdC, Costenaro F, Scaffaro LA, Farenzena M, Gastaldo F and Czepielewski MA (2023) Enhancing Cushing’s disease diagnosis: exploring the impact of desmopressin on ACTH gradient during BIPSS. Front. Endocrinol. 14:1224001. doi: 10.3389/fendo.2023.1224001 Received: 17 May 2023; Accepted: 11 July 2023; Published: 03 August 2023. Edited by: Fabienne Langlois, Centre Hospitalier Universitaire de Sherbrooke, Canada Reviewed by: Filippo Ceccato, University of Padua, Italy Matthieu St-Jean, Université de Sherbrooke, Canada Copyright © 2023 Almeida, Rodrigues, Costenaro, Scaffaro, Farenzena, Gastaldo and Czepielewski. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Tobias Skrebsky de Almeida, tsalmeid@gmail.com Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fendo.2023.1224001/full

Background: Café-au-lait skin macules, Cushing syndrome (CS), hyperthyroidism, and liver and cardiac dysfunction are presenting features of neonatal McCune–Albright syndrome (MAS), CS being the rarest endocrine feature. Although spontaneous resolution of hypercortisolism has been reported, outcome is usually unfavorable. While a unified approach to diagnosis, treatment, and follow-up is lacking, herein successful treatment and long-term follow-up of a rare case is presented. Clinical case: An 11-day-old girl born small for gestational age presented with deterioration of well-being and weight loss. Large hyperpigmented macules on the trunk, hypertension, hyponatremia, hyperglycemia, and elevated liver enzymes were noted. ACTH-independent CS due to MAS was diagnosed. Although metyrapone (300 mg/m2/day) was started on the 25th day, complete remission could not be achieved despite increasing the dose up to 1,850 mg/m2/day. At 9 months, right total and left three-quarters adrenalectomy was performed. Cortisol decreased substantially, ACTH remained suppressed, rapid tapering of hydrocortisone to physiological dose was not tolerated, and supraphysiological doses were required for 2 months. GNAS analysis from the adrenal tissue showed a pathogenic heterozygous mutation. During 34 months of follow-up, in addition to CS due to MAS, fibrous dysplasia, hypophosphatemic rickets, and peripheral precocious puberty were detected. She is still regularly screened for other endocrinopathies. Conclusion: Neonatal CS due to MAS is extremely rare. Although there is no specific guideline for diagnosis, treatment, or follow-up, addressing side effects and identifying treatment outcomes will improve quality of life and survival. Introduction McCune–Albright syndrome (MAS) is a rare mosaic disorder of remarkable complexity with an estimated prevalence of 1/100,000 and 1/1,000,000 (1). Timing of postzygotic missense gain of function mutation of GNAS encoding stimulatory Gαs determines the extent of tissue involvement, imposing a unique clinical phenotype. Although a combination of two or more classical features, such as fibrous dysplasia of bone (FD), café-au-lait skin macules, and hyperfunctioning endocrinopathies (gonadotropin-independent gonadal function, nonautoimmune hyperthyroidism, growth hormone excess, and neonatal hypercortisolism), are diagnostic, renal, hepatobiliary, and cardiac involvement have also been reported (2–4). Adrenocorticotropic hormone (ACTH)-independent adrenal Gαs activation results in the rarest endocrine feature of MAS, which almost invariably presents in the neonatal period: Cushing syndrome (CS). Due to greater burden of Gαs-mutation-bearing cells, the presence of CS is correlated with increased number of accompanying features of MAS and a poorer outcome. Although there is spontaneous resolution in 33% of cases with neonatal CS, mortality occurs with a high rate of 20% (4). A dilemma for the clinician is that most publications to date have been case reports, and there is as yet no guideline for diagnosis, treatment, or follow-up. Here, a rare case of severe CS due to MAS, underlining the unique clinical phenotype specific to the neonatal period, is presented. Our goal is to offer a practical approach based on 3 years of clinical experience of this rare disorder that will help navigate challenges during follow-up. Case presentation A baby girl, born small for gestational age with a birthweight of 2,340 g (−2.1 SDS) and a head circumference of 32.6 cm (−1.61 SDS) was admitted to the neonatal intensive care unit in the first day of life for respiratory distress. She was the second child of a healthy non-consanguineous Caucasian couple, born 38 weeks of gestation via cesarean section following an uneventful pregnancy. Alanine aminotransferase [ALT, 2,376 U/L (normal, 0–40)] and aspartate aminotransferase [AST, 875 U/L (normal, 0–40)] were elevated; gamma-glutamyl transferase and bilirubin were normal. Antibiotics were administered intravenously after a diagnosis of possible neonatal sepsis. Respiratory distress resolved, and liver enzymes decreased (ALT, 687 U/L; AST, 108 U/L). As soon as the antimicrobial treatment was completed, she was discharged in the seventh day of life. She was referred to our center, 4 days later, for failure to thrive (2,315 g), difficulty in feeding, and deterioration of general health. On physical examination, round facies, elongated philtrum and retro-micrognatia, hyperpigmented macules both at the front and back of the trunk and on labia majora, which do not cross midline, and hypertrichosis on the forehead and extremities were noted (Supplementary Figure S1). Newborn reflexes were hypoactive, blood pressure was 100/70 mmHg, and second-degree cardiac murmur was also detected. Systems were normal otherwise. Laboratory findings revealed hyponatremia, impaired renal and liver function tests, tubulopathy, and proteinuria, while blood count was normal (hemoglobin, 10.4 g/dl; leukocyte, 25.0 × 103/μl; platelet count, 449×103/μl) (Table 1). Hyponatremia resolved with fluid treatment, while liver enzymes, blood urea nitrogen, and creatinine remained elevated. Further endocrine evaluation revealed an elevated serum basal cortisol [225.68 g/dl (N, 6.7–22.6 µg/dL)] and 24-h urinary free cortisol [1,129 μg/day (N, 1.4–20 μg/day)]. Serum cortisol was not suppressed during overnight high-dose dexamethasone suppression test (Table 2) (5). Thyroid hormones were consistent with non-thyroidal illness. Table 1 Table 1 Laboratory investigations on admission, prior to medical treatment (19 days), after medical treatment (6 months), and post-adrenalectomy. Table 2 Table 2 Endocrine evaluation prior to medical treatment (19 days), after medical treatment (6 months), and post-adrenalectomy. ACTH-independent CS and café-au-lait spots suggested MAS. Hypercortisolism-related complications emerged. On the 11th day, hyperglycemia (blood glucose, 250 mg/dl) was seen, and it persisted after cessation of intravenous fluids in the exclusively breastfed neonate; thus, 0.5 U subcutaneous neutral protamine Hagedorn insulin (NPH) (three times a day) was initiated on the 16th day of life when blood glucose was 340 mg/dl, and serum insulin was 18.10 μIU/ml. Hypertension (110/90 mmHg) and hypokalemia were triggered by mineralocorticoid action of excessive cortisol on 20th day. Spironolactone (2 mg/kg/day) was started, and nifedipine (0.5 mg/kg/day) was added in order to control blood pressure (Supplementary Figure S2). Since immunosuppressive effects of excess cortisol may increase the risk for opportunistic infections, Pneumocystis jirovecii prophylaxis was started and live vaccines were postponed. Features of MAS and accompanying hyperfunctioning endocrinopathies were screened (Table 2). On ultrasonography, adrenal glands were hypertrophic; kidneys showed increased parenchymal echogenicity, loss of separation between the cortex and medulla, and enhanced medullary echogenicity; and size and echogenicity of the liver were normal. Magnetic resonance imaging of the abdomen confirmed that adrenal glands were hypertrophic (right and left adrenal gland were 24×22×18 mm and 18×19×20 mm in size, respectively) and lobulated. Echocardiogram revealed left ventricular hypertrophy. Bone survey verified generalized decrease in bone mass and revealed areas of irregular ossification and radiolucency in radius, ulna, and distal tibia, which were interpreted as osteoporosis due to hypercortisolism (Supplementary Figure S1). Medical treatment Metyrapone (300 mg/m2/day, per oral, in four doses) was started on the 25th day (Supplementary Figure S2) (6). Since liver function tests were impaired, metyrapone was preferred over ketoconazole. Soon after metyrapone was started, hyperglycemia and hypertension improved, enabling the discontinuation of insulin and nifedipine. Spironolactone was also gradually tapered and discontinued after 13 days of metyrapone treatment, and she was discharged. The dose of metyrapone was adjusted frequently, according to clinical findings and serum cortisol levels during regular visits. However, even after gradually increasing metyrapone dose to 1,850 mg/m2/day over the course of 6 months, total biochemical suppression of serum cortisol could not be achieved (Supplementary Figure S3A), and the patient had progressive loss of bone mineral density, persistent left ventricular hypertrophy, and a lack of catch-up growth. In addition to that, café-au-lait macules became darker, dehydroepiandrosterone sulfate (DHEA-S) gradually increased (Table 2), and previously non-existent marked clitoromegaly was noted as a side effect of high-dose metyrapone. She was also prescribed ursodeoxycholic acid (15 mg/kg/day); however, liver enzymes remained high (Table 1). Right total and left three-quarters adrenalectomy Right total and left three-quarters adrenalectomy was carried out at 9 months of age in light of the patient’s continued clinical findings of hypercortisolism, the existence of unfavorable prognostic markers (high cortisol levels upon admission and heart and liver problems), and the adverse effects of high-dose metyrapone. The patient was administered 100 mg/m2/day glucocorticoids (GC) perioperatively; however, she developed symptoms of adrenal insufficiency. The required GC dose to attain euglycemia, restore general well-being, and resolve adrenal insufficiency was 300 mg/m2/day. Fludrocortisone (0.05 mg/day) was also started. Following surgery, supraphysiological doses of GC were required, as she suffered frequent symptoms of adrenal insufficiency (hypoglycemia, malaise, and loss of appetite). GC dose could be tapered very slowly, and a daily dose of 15 mg/m2/day could be attained in 2 months. As liver function tests, serum cortisol levels and left ventricular hypertrophy all improved following adrenalectomy (Table 1). Bilateral nodular adrenal hyperplasia was observed in the pathological evaluation of surgical specimen, while the findings of liver wedge biopsy were non-specific (Supplementary Figure S4). Sequence analysis of GNAS from the surgical sample of adrenal gland revealed a heterozygous, previously described missense mutation in exon 8 (c.2530C>A, p.Arg844Ser), while the sequence analysis of the GNAS gene from peripheral blood sample was normal. Lymphocyte activation was normal 3 months post-adrenalectomy, and immunization schedule for live vaccines was established. Other findings of MAS She had breast development and vaginal bleeding that lasted 2 days when she was 7 months old, which repeated five more times after the adrenalectomy till 26 months of age. Breast development was Tanner stage 3, and bone age was markedly advanced (4 years and 2 months), despite severe hypercortisolism. On pelvic ultrasonography, uterus was enlarged to 34×22×24 mm; thus, letrozole (0.625 mg, per oral) was started at 26 months of age. She also developed marked hypophosphatemia at the age of 6 months (Table 1). Radiological investigations since birth demonstrated severe osteopenia and lytic lesions, which were attributed to severe hypercortisolism; however, overt lesions of FD were not confirmed. When she was 9 months old, FGF-23 was elevated [122 pg/ml (normal <52)], which suggested hypophosphatemic rickets associated with FD. Oral phosphate (8 mg/kg) and calcitriol (18 ng/kg) were started. At the age of 23 months, bone survey revealed sclerosis of the base of the skull and maxilla and FD in the lower extremities. She has been on oral phosphate (58.7 mg/kg/day), while calcitriol was ceased. She is now 34 months old with severe short stature [height, 81 cm (−3.5 SDS); weight, 9,580 g (−3.7SDS)] (Supplementary Figure S3B). She had been under regular clinic visits and has been on 15 mg/m2/day hydrocortisone and fludrocortisone 0.025 mg/day, letrozole (1×6.25 mg/day), phosphate (58 mg/kg), and ursodeoxycholic acid (100 mg/day) (Supplementary Figure S2). She has six words, cannot form two-word sentences, shows body parts, cannot stand up from supine position without support, and takes a few steps with support. Despite regular physiotherapy and ergotherapy, developmental delay is evident (Bayley Scales of Infant and Toddler Development III language scale, 13/79; motor scale, 2/46). Discussion ACTH-independent CS and café-au-lait macules suggested MAS in this case. Interestingly, this patient was admitted for hyponatremia and hyperglycemia requiring insulin treatment. Neonatal MAS and CS are rare conditions, and presentation of this case is quite unique (4). The earlier the timing of somatic mutation, the greater the burden of Gsα-mutation-bearing cells leading to widespread tissue involvement in MAS. In the current case, adrenal, hepatic, cardiac, renal, and bone tissue involvement were evident in first weeks of life, while precocious puberty and hypophosphatemic rickets were observed later. A lifetime risk of additional tissue involvement is being acknowledged. CS is the rarest endocrine manifestation of MAS, which appears in <5%–7.1%. It presents exclusively within the first year of life (median age, 3.1 months) where features may develop as early as in utero (2–4, 7). The fact that our case was SGA and had moon facies and hirsutism with impaired linear growth, weight gain, hyperglycemia, hypertension, and nephrocalcinosis detected in the neonatal period, suggested severe, in utero onset CS. Upon suspicion, both comorbidities (hyperthyroidism, excess growth hormone, FD, and cardiac and hepatobiliary function) of MAS and complications of GC excess (hypertension, hyperglycemia, hyperlipidemia, nephrocalcinosis, decreased bone mineral density, and muscle atrophy) were assessed (1, 3). Since the initial description of MAS, only 20 neonates with CS have been described with various initial basal serum cortisol ranging from 9.6 to 80.1 µg/dl, and data regarding long-term follow-up and outcome are still developing (1, 2, 8–11). Disease course is heterogenous, and spontaneous resolution of hypercortisolism has been reported (30%) since Gs-bearing cells are mostly located in the fetal adrenal zone, which normally undergoes apoptosis after birth. However, the outcome is mostly unfavorable in cases with extensive endocrine and extra-endocrine manifestations (1, 2, 8–15). Brown et al. reported poorer prognosis and a lower likelihood of spontaneous remission of adrenal disease in patients with cardiac (cardiomyopathy) and liver involvement (hepatocellular adenomas, inflammatory adenomas, choledochal cysts, neonatal cholestasis, and hepatoblastoma). It was hypothesized that these patients have a greater burden of Gsα mutation (3, 4). Treatment of neonatal CS is a long and challenging path where both cortisol excess and its complications should be targeted. Marked hypercortisolism that precipitate neonatal diabetes requiring insulin treatment like our patient is rare and was previously reported only in six patients with CS (4). Until hypercortisolism is managed, hyperglycemia should be treated with insulin. Hypertension is due to mineralocorticoid effect of excess cortisol; thus, blood pressure lowering agents of choice should be aldosterone antagonists (spironolactone) or potassium-sparing diuretics. The treatment strategy of hypercortisolism is determined by disease severity. In a mildly affected case, medical treatment with an expectation of spontaneous resolution (due to previously stated apoptosis of fetal adrenal zone) may be of choice (3, 4, 16–19). Metyrapone, ketoconazole, and mitotane are medical options for lowering cortisol (20–23). Since our patient had impaired liver function, metyrapone, a potent, rapid acting relatively selective inhibitor of 11-hydroxylase was preferred over ketoconazole for its low risk of hepatotoxicity. Reports reviewing adult data suggest an initial dose of 500–750 mg/day and achievement of biochemical control with 1,500 mg/day (23). However, the initial and maximum dose of metyrapone in neonates is unclear; some authors recommend 300 mg/m2/day in four equal doses (6). In our case, adequate biochemical and clinical suppression of cortisol with metyrapone was not achieved despite an increase in dose from 300 to 1,850 mg/m2/day. There are important issues to be considered while using a steroidogenesis inhibitor like metyrapone. Monitoring biochemical response is essential, not only for dose titration and management of cortisol excess but also for adrenal insufficiency due to possible overtreatment. Clinical signs of adrenal insufficiency should always be questioned and assessed. The 24-h urinary free cortisol is the commonly used method; however, it may be impractical due to difficulties in the collection of urine in infants. Alternative methods may be the measurement of early morning serum cortisol and ACTH (23). Low ACTH level may indicate hypercortisolism or may be a sign of suppression due to long-term exposure to hypercortisolism. However, there are deadlocks to be considered in the evaluation of these measurements. A high cortisol level measured by immunoassays does not always indicate an actual elevation. It should be kept in mind that cortisol immunoassays exhibit significant cross-reactivity with cortisol precursors that may be elevated in patients treated with a steroidogenesis inhibitor (especially with metyrapone, which is known to increase 11-deoxycortisol). Such cross-reactivity can be a cause for overestimation of cortisol and may lead to risk of overtreatment (24, 25). It has been suggested that the patients on metyrapone should be biochemically monitored via specific methods, such as mass spectrometry (24–26). Metyrapone is a relatively selective inhibitor of 11-hydroxylase and 18-hydroxylase. Recent in vitro studies indicate greater inhibitory action of metyrapone on aldosterone synthase, resulting in significant reversible reduction in both cortisol and aldosterone. The loss of negative feedback leads to an increase in ACTH, which causes an accumulation of cortisol and aldosterone precursors resulting in an increase in adrenal androgens (23). Although we could not serologically prove an increase in ACTH, hyperpigmentation and the increase in adrenal androgens confirm this mechanism. As far as we know, an increase in DHEA-S causing virilization was an unreported side effect of metyrapone. Clinical (clitoromegaly and hirsutism) and laboratory (DHEA-S) signs of hyperandrogenism should be monitored when higher doses of metyrapone are required. In the severely affected case with CS, where medical treatment is inadequate and the chance of spontaneous resolution is subsiding, adrenalectomy is indicated when medically feasible. Brown et al. suggested that the presence of comorbid cardiac and liver disease like in our case should prompt consideration for early adrenalectomy (4). Although a previous correlation with initial serum cortisol level and prognosis was not established, it may be speculated that excessively high serum cortisol level is associated with increased number of Gsα-mutation-bearing adrenal cells. Thus, we suggest that in neonatal CS due to MAS, initial very high serum cortisol levels, like our case, may be a negative prognostic factor both for spontaneous resolution and clinical response to medical treatment. In infants with severe CS, bilateral adrenalectomy is generally performed. Alternatives like unilateral adrenalectomy and one-side total, other-side three-quarters adrenalectomy may be considered to avoid the requirement for lifelong GC and mineralocorticoid replacement. Unilateral adrenalectomy was reported to successfully improve clinical symptoms and endocrinological status in adult studies; nevertheless, recurrence during follow-up was 23.1%, while 17.5% required contralateral adrenalectomy (27–29). Since the causes of CS in adult series are variable and different from pediatric CS due to MAS, it should be borne in mind that reproducibility of adult data is poor. In CS due to MAS, Gsα-mutation-bearing adrenal gland cells are heterogeneously distributed, and partial adrenalectomy may carry the risk of inadequate management and recurrence. Only a few pediatric case reports addressed this issue. Unilateral adrenalectomy of the larger gland was performed in two neonates with CS due to MAS; remission was achieved for 2 years (30, 31). Itonaga et al. reported a 6-month-old neonate with MAS-associated CS treated with right-sided total adrenalectomy and left-sided half adrenalectomy with remission for 2 years (32). Although these cases were less severe [basal serum cortisol: 16.9, 18.5, and 23.4 µg/dl, respectively (N: 6.2–18.0 µg/dL)], we preferred to perform partial adrenalectomy (right total and left three-quarters adrenalectomy) and succeeded. Our patient has been in remission for more than 2 years. In the largest case–control analysis of CS in patients with MAS, overall mortality was 20% (six cases) where four of them were deceased following bilateral adrenalectomy (66.7% of all deaths) (4). Anaphylaxis (or adrenal insufficiency), sudden cardiac arrest, sepsis, and sudden death were listed as causes of mortality in those four cases where GC dose and process of GC tapering were not clearly described. The fact that our patient required high-dose GC during peri- and postoperative period to restore well-being, tapering to maintenance dose was very slow, and she is still on maintenance dose GC, suggests that rapid tapering of GCs should be avoided and, although being speculative, may explain sudden death following adrenalectomy. Gross motor developmental delay may be caused by prenatal exposure to excess GCs. Prenatal GC treatment for possible congenital adrenal hyperplasia or risk of premature birth have been shown to result in cognitive deficits after birth. Furthermore, children who develop CS later in life may experience a decline in cognitive and school performance where the younger the age of onset, the greater the deterioration in IQ scores (3, 4, 33, 34). Since transgenic mice with Gsα mutation was shown to have short- and long-term memory deficits and impaired associative and spatial learning, it may also be speculated that Gsα mutation may also be present in the central nervous system (35, 36). The establishment of diagnosis of FD follows a characteristic and predictable time course. Although GNAS mutations are acquired early in embryogenesis, skeletal development appears to be relatively normal in utero, without frank clinical signs of FD at birth. Boyce et al. affirmed that FD lesions become apparent over the first several years of life and expand during childhood and adolescence, like our case. Previous case reports have also stated severe osteoporosis, rickets, polyostotic irregular lucencies, pathological fractures, and biopsy-proven FD during infancy (1, 2, 8–15). The exact pathophysiological mechanism is unclear, and Gsα activation in abnormally differentiated osteocytes is accused. FGF-23 overproduction is an inherent feature of FD, and most patients have elevated circulating levels of FGF-23, but frank hypophosphatemia is rare. The increase in FGF-23 is linked to substantial skeletal involvement. Although FGF-23 levels may wax and wane over time, an increase in FGF-23 usually occurs during periods of rapid growth like infancy and adolescence. Concurrent hyperfunctioning endocrinopathies like hyperthyroidism or CS may also adversely affect bone health. Peripheral precocious puberty (PP) is the most frequent presenting feature in female patients with MAS (85%) (6). To date, a safe, effective, and long-term treatment for PP in girls with MAS has not been established. The benefits of current interventions on the ultimate outcome of interest, adult height, have not been well-established due to the rarity of the condition and heterogeneous nature of the disease. Despite the small sample size, studies have concluded that letrozole resulted in a statistically significant decrease in the bone age/chronological age ratio, growth velocity, hence increasing predicted adult height (37). Growth outcome in MAS is not only dependent on timing of pubertal onset but on several other disease components (skeletal involvement and endocrinopathies) as well. Hyperthyroidism and growth hormone excess may accelerate growth, while CS may decelerate it (37, 38). Lack of consensus on both medical and surgical treatment strategies were major obstacles while navigating this case of severe neonatal MAS. The eminence of this report is that it presents current literature with clinical experience on this rare case of neonatal CS due to MAS. High index of suspicion for MAS in a neonate with extensive café-au-lait macules and symptoms of hypercortisolism is the key for early recognition and intervention. Initial excessive cortisol in neonatal CS may be a negative prognostic factor for spontaneous resolution and response to medical treatment, indicating early right total and left three-quarters adrenalectomy. Post-adrenalectomy survival may be related to close supervision during GC tapering. Data availability statement The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material. Ethics statement Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article. Author contributions YU collected and analyzed data, drafted the initial manuscript, and reviewed and revised the manuscript. OG collected data. İU, HH, BG, SE, and TK collected data and reviewed and revised the manuscript. ZO and EG analyzed data, conceptualized the work, and revised and critically reviewed the manuscript for important intellectual and medical content. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. Acknowledgments We thank our patient’s family for providing consent for publication of this work. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2023.1209189/full#supplementary-material Supplementary Figure 1 | (A) The findings of physical and radiologic examination. Notice cushingoid facies, hyperpigmented macules that does not cross the midline at the front of the trunk. (B) Anteroposterior radiographs reveal irregularities in radius, ulna and femur. Although generalized osteopenia improves at 34 months, FD lesions become prominent over months. Supplementary Figure 2 | Timeline of the course of symptoms in neonatal McCune Albright Syndrome noting adjustments made in treatment. Grey box denotes age in days for the first month of life then in months. NPH: Neutral Protamine Hagedorn insulin, CS: Cushing syndrome, PP: precocious puberty. Supplementary Figure 3 | (A) Change in serum cortisol with increased metyrapone (methyrapone was initiated on day 25). (B) Growth chart, the arrow represents right total and left three quarters adrenalectomy. Supplementary Figure 4 | Representative histological features of nodular adrenal hyperplasia. (A, show low-power while (C) Show high-power views. References 1. Lourenço R, Dias P, Gouveia R, Sousa AB, Oliveira G. Neonatal McCune-Albright syndrome with systemic involvement: a case report. J Med Case Rep (2015) 9:189. doi: 10.1186/s13256-015-0689-2 PubMed Abstract | CrossRef Full Text | Google Scholar 2. Corsi A, Cherman N, Donaldson DL, Robey PG, Collins MT, Riminucci M. Neonatal McCune-Albright syndrome: A unique syndromic profile with an unfavorable outcome. JBMR Plus (2019) 3:e10134. doi: 10.1002/jbm4.10134 PubMed Abstract | CrossRef Full Text | Google Scholar 3. Boyce AM, Collins MT. Fibrous dysplasia/McCune-Albright syndrome: A rare, mosaic disease of Gα s activation. 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(2018) 7(12):1424–31. doi: 10.1530/EC-18-0344 PubMed Abstract | CrossRef Full Text | Google Scholar Keywords: McCune Albright syndrome, neonatal Cushing syndrome, metyrapone, adrenalectomy, follow-up Citation: Unsal Y, Gozmen O, User İR, Hızarcıoglu H, Gulhan B, Ekinci S, Karagoz T, Ozon ZA and Gonc EN (2023) Case Report: Severe McCune–Albright syndrome presenting with neonatal Cushing syndrome: navigating through clinical obstacles. Front. Endocrinol. 14:1209189. doi: 10.3389/fendo.2023.1209189 Received: 20 April 2023; Accepted: 04 July 2023; Published: 25 July 2023. Edited by: Martin Oswald Savage, Queen Mary University of London, United Kingdom Reviewed by: Li Chan, Queen Mary University of London, United Kingdom Sasha R Howard, Queen Mary University of London, United Kingdom Tomoyo Itonaga, Oita University, Japan Copyright © 2023 Unsal, Gozmen, User, Hızarcıoglu, Gulhan, Ekinci, Karagoz, Ozon and Gonc. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. *Correspondence: Yagmur Unsal, yagmurunsal@yahoo.com Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. From https://www.frontiersin.org/articles/10.3389/fendo.2023.1209189/full

Abstract Introduction and importance Pheochromocytoma and Cushing's syndrome are rare endocrine conditions caused by tumors in the adrenal gland. These conditions are classified under Multiple Endocrine Neoplasia (MEN) syndrome, characterized by the development of multiple tumors in the endocrine system. However, diagnosing these conditions can be challenging as they often lack clear symptoms, requiring careful evaluation, monitoring, and treatment to prevent complications. Case presentation A 23-year-old male recently presented with right-sided abdominal fullness and lipoma-like masses on the torso. Over a span of six months, the abdominal mass nearly doubled in size, accompanied by elevated levels of catecholamines, cortisol, parathyroid hormone (PTH), and calcitonin. Surprisingly, the patient remained asymptomatic despite these abnormal lab values. CT imaging revealed a substantial increase in the size of the mass in the right adrenal gland, from 6 × 7 cm to approximately 11.2 × 10.2 × 9 cm. Clinical discussion Pheochromocytoma secretes catecholamines and often leads to hypertension and related symptoms. Interestingly, most individuals with pheochromocytoma do not exhibit obvious symptoms, necessitating blood and urine tests, along with imaging studies, for accurate diagnosis. The size of the tumor does not necessarily indicate the severity of symptoms. MEN-2, a genetic syndrome, is characterized by pheochromocytoma, medullary thyroid carcinoma, and hyperparathyroidism. Additionally, methods for diagnosing Cushing's syndrome, caused by excess cortisol production, are discussed. Conclusion Early diagnosis and genetic counseling are crucial in preventing complications associated with these conditions. By identifying them, appropriate treatment can be ensured for positive outcomes of patients and their families. Previous article in issue Next article in issue Keywords Pheochromocytoma Multiple Endocrine Neoplasia (MEN) syndrome Cushing's syndrome Rare Case Report Abbreviations CT computed tomography MRI Magnetic resonance imaging USG Ultrasonography 131I-MIBG iodine 131 labeled meta-iodobenzylganidine RAAS Renin-angiotensin-aldosterone system 1. Introduction Pheochromocytoma are catecholamine secreting tumors of chromaffin cells of adrenal medulla. It can be found anywhere in the body, with the majority being intra-abdominal and those other than adrenal medulla are referred to as paragangliomas [1,2]. Pheochromocytoma typically secretes norepinephrine and epinephrine, with norepinephrine being the primary catecholamine. However, some tumors may only secrete one of the two, and rarely, some may secrete dopamine or dopa [3]. Vast majority >90 % of adrenal neoplasms are benign non-functional adenomas [4].About 10 % of pheochromocytomas are malignant and 10 % of cases are found on both sides. Additionally, approximately 40 % of pheochromocytomas are caused by genetic factors and can be associated with inherited syndromes [5]. Pheochromocytoma is found to be associated with MEN-2. MEN-2 is a hereditary genetic condition that is caused by a de novo mutation in the RET gene. It is inherited in an autosomal dominant fashion and is mainly characterized by medullary thyroid carcinoma, pheochromocytoma and parathyroid adenoma or hyperplasia [6]. MEN syndrome can be MEN-1, MEN-2A and MEN-2B. MEN-1 is characterized by pituitary tumors (prolactin or growth hormone), pancreatic endocrine tumors and parathyroid adenomas. Additionally, other tumors such as foregut carcinoids, adrenocortical adenomas, meningioma, lipomas, angiofibromas and collagenomas may also occur in MEN-1. MEN-2A is characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid adenoma/hyperplasia; it can also be associated with cutaneous lichen amyloidosis and Hirschsprung disease. On the other hand, MEN-2B is characterized by familial medullary thyroid cancer, pheochromocytoma, mucosal neuromas, gastrointestinal tract issues, musculoskeletal and spinal problems. [7]. Cushing syndrome results from hypercortisolism and is characterized by hypertension, weight gain, easy bruising, and central obesity [4]. Cushing's disease refers to ACTH-dependent cortisol excess caused by a pituitary adenoma, while ACTH-independent cortisol excess due to non-pituitary causes such as excess use of glucocorticoids, adrenal adenoma, hyperplasia, or carcinoma is referred to as Cushing syndrome [8]. This case report has been written according to the SCARE checklist [9]. 2. Case presentation A 23-year-old male presented to our surgery department with the chief complaint of right sided abdominal fullness for six months. According to the patient a mass was incidentally reported six months back while he was under-evaluation for mild trauma due to road traffic accident. Six months back, the mass was approximately 6 × 7 cm, while at the time of presentation to our department the mass was approximately 11.2 × 10.2 × 9 cm (CT abdomen) which was globular in shape, had regular margin, and moved with respiration. He had no history of hypertension, headache, palpitation, sweating, pallor, recent weight loss, abdominal pain, psychological disturbance, dizziness, loss of consciousness, dark color urine, burning micturition, had normal bowel and bladder habit. Past history and family history were insignificant. He was not under any long-term medication and no known drug allergies. He occasionally smokes and consumes alcohol. On physical examination at the time of presentation, multiple soft, mobile, painless, subcutaneous nodules like lipoma were present over the torso. His height was 176.8 cm, weight 68 kg, BMI 21.8 kg/m2 (body mass index). He had blood pressure of 110/70 mm of Hg taken in left arm at sitting position, heart rate of 62 beats/min, respiratory rate of 24/min, temperature of 96.6 °F, SPO2 of 98 % at right hand. A mass was palpable on the right side of abdomen, otherwise abdomen was soft, non-tender, normal bowel sound was present. Chest, cardiac and neurologic examinations were all normal. Initial laboratory evaluation revealed 24 h. urine metanephrine of 5415 μg/24 h (normal: 25–312 μg/24 h.); 24 h. urine VMA of 32.2 mg/24 h. (normal: <13.60 mg/24 h.); serum cortisol of 535.16 nmol/l after overnight low dose dexamethasone(1 mg) suppression test (normal: <50 nmol/l);24 h. Urine free cortisol of 526.61 nmol/24 h. (normal: 30–145 nmol/24 h) PTH(intact) of 89.2 pg./ml (normal: 15–65 pg./ml); serum calcitonin of 15.2 pg./ml (normal: ≤8.4 pg./ml); serum CEA of 4.72 ng/ml (normal: 0.0–4.4 ng/ml); serum DHEA of 1.19 ng/ml (normal: 1.7–6.1 ng/ml). Baseline investigation: Hematology, urine routine/microscopic, electrolytes were within the normal range. Additional laboratory findings were as in the Table 1. Table 1. Lab evaluation Result Reference Unit Metanephrine, urine 24 h 5415 25–312 μg/24 h VMA, urine 24 h 32.2 <13.60 mg/24 h VMA, urine 12.88 – ng/l Cortisol, serum, overnight DST 535.16 <50 nmol/l Cortisol, urine 24 h 526.61 30–145 nmol/24 h ACTH, complete 28.3 7.2–63.3 pg/ml DHEA, serum 1.19 1.7–6.1 ng/ml CEA, serum 4.72 0.0–4.4 ng/ml Phosphorus, serum 3.0 2.5–4.5 mg/dl Albumin, serum 5.2 3.5–5.2 g/dl Calcitonin, serum 15.2 ≤8.4 pg/ml Calcium, serum 8.94 8.6–10.0 mg/dl PTH (intact) 89.2 15–65 pg/ml aldosterone 8.7 7.0–30 g/dl Plasma rennin activity 1.42 0.10–6.56 ng/ml/h Aldosterone-rennin ratio 6.13 ≤20 Creatinine, urine 36 – mg/dl DST - dexamethasone suppression test; VMA - vanilmandelic acid; ACTH - adrenocorticotropic hormone; DHEA - dehydroepiandrosterone; CEA - carcino-embryonic-antigen; PTH - parathyroid hormone. 2.1. USG abdomen USG abdomen (Fig. 1, Fig. 2) showed well defined mixed echoic area in Right adrenal region measuring 12.7 × 10.7 cm in size. There was presence of internal vascularity with multiple foci of cystic compound. The lesion displaced the right kidney inferiorly. Download : Download high-res image (102KB) Download : Download full-size image Fig. 1. USG abdomen. Download : Download high-res image (106KB) Download : Download full-size image Fig. 2. USG abdomen. 2.2. Plane and contrast CT scan of abdomen Plane and contrast CT scan of Abdomen (Fig. 3) showed approximately 11.2 × 10.2 × 9 cm sized, relatively well defined heterogeneous soft tissue density lesion with well-defined enhancing wall in right adrenal region. Non-enhancing areas were noted within the mass suggestive of necrosis. Few calcific foci were noted within the mass with no obvious hemorrhagic component. The lesion showed heterogeneous enhancement post contrast image. Download : Download high-res image (192KB) Download : Download full-size image Fig. 3. CT abdomen. After all the workup patient was given diagnosis of right sided Pheochromocytoma associated with MEN syndrome, with ACTH-independent Cushing's syndrome and right adrenalectomy was performed. 2.3. Pathology report 2.3.1. Gross descriptions The specimen was globular mass measuring 14.5 × 10 cm, with smooth outer surface. On sectioning, the mass was well circumscribed, soft and yellow-brown, predominantly solid with cyst formation. The size of cyst ranges from 0.3 to 3.5 cm in diameter. Areas of hemorrhages were noted. 2.3.2. Microscopic description Section showed tumor cells arranged in well-defined nests (Zellballen), alveolar and diffuse pattern with intervening fibrovascular stroma. The cells were intermediate to large sized, polygonal with finely granular amphophilic cytoplasm. The nuclei showed mild to moderate pleomorphism and were round to ovoid, with prominent nuclei noted. No capsular invasion, vascular invasion and necrosis. Areas of hemorrhage were seen. Mitosis 0–1/10 high power field was noted (Figs. 4 and 5). Download : Download high-res image (1MB) Download : Download full-size image Fig.a Diffuse Zellbalen pattern with intervening fibrous stroma. Fig.b Mild to moderate pleomorphic nuclei with abundant hemorrhage. Fig.c Low power field with intact capsule. Download : Download high-res image (352KB) Download : Download full-size image Figs. 4 and 5. Fig. 4 Intra-operative resection of tumor; Fig. 5 tumor after resection. 3. Discussion In Pheochromocytoma activation of the alpha-one adrenergic receptor by catecholamine in the vascular bed causes vasoconstriction and leads to a rise in blood pressure. Similarly, activation of the beta-one receptor in the heart enhances the chronotropic and inotropic effect of the myocardium, leading to an increase in heart rate and cardiac output. In addition, activation of the beta-one receptor in the juxtaglomerular cells of the kidney activates the RAAS system. These receptor activation result in cardiovascular and sympathetic changes, such as hypertension, palpitation, headache, sweating, trembling, and anxiety [10]. In Pheochromocytoma, the patient may have a 10-fold increase in plasma catecholamines, but the hemodynamic response can still fall within the normal range due to desensitization of the cardiovascular system. When catecholamine levels are elevated for a prolonged period, the alpha-one receptors in blood vessels may be down-regulated, making norepinephrine unresponsive in raising peripheral vascular resistance, which can lead to normal blood pressure. Similarly, a marked decrease in beta-one receptors in the heart could explain the normal heart rate, which was observed in our asymptomatic patient with Pheochromocytoma [11]. Sometimes in asymptomatic patients, the size of the tumor tends to be larger than in those with hyperfunctioning tumors [12]. However, medical interventions such as surgery, anesthesia induction, intravenous urography contrast, or manipulation of the tumor can trigger adrenergic and hypertensive crises, so biopsy is usually contraindicated in pheochromocytoma [13]. The diagnosis of pheochromocytoma is typically based on measuring plasma and urinary levels of catecholamines and their derivatives such as metanephrine and vanillylmandelic acid. The most reliable test is the measurement of urinary metanephrine as its excretion levels are relatively higher [13,14]. The combination of 131I-MIBG scintigraphy along with diagnostic urinary and blood tests can further enhance the sensitivity of the test. Specifically, the urinary normetanephrine test is considered the most sensitive single test for detecting Pheochromocytoma [15,16]. In addition to a 24-h urine test and blood test, if the lab results are positive for Pheochromocytoma or paragangliomas, further diagnostic tests may be recommended, such as a CT scan, MRI, m-iodobenzylganidine (MIBG) imaging, or positron emission tomography (PET) [16,17]. In our patient 24 h. urine metanephrine of 5415 μg/24 h (normal: 25–312 μg/24 h.); 24 h. urine VMA of 32.2 mg/24 h. (normal: <13.60 mg/24 h.) and imaging confirmation of right adrenal mass lead to the diagnosis of right sided pheochromocytoma. Our patient with pheochromocytoma was tested for parathyroid hormone and calcitonin due to the association of pheochromocytoma with MEN-2 [18]. MEN-2 can be diagnosed biochemically by measuring the baseline levels of calcitonin, parathyroid hormone and serum calcium along with blood tests for catecholamines and their metabolites to detect pheochromocytoma [19]. In our patient, multiple soft, mobile, painless, subcutaneous nodules like lipoma were present over the torso(MEN-1) and high levels of parathyroid hormone and calcitonin were detected(MEN-2). These findings can be correlated with MEN syndrome. USG of the neck revealed no abnormalities of thyroid and parathyroid gland in our patient so prophylactic thyroidectomy was not done, instead he was counseled for follow up if any symptoms or thyroid swelling appears. The diagnosis of Cushing's syndrome typically involves measuring the levels of 24-h urine free cortisol and assessing the suppression of cortisol in response to a 1 mg overnight dexamethasone test. If cortisol levels remain elevated despite the test, the next step is to measure serum ACTH levels. If ACTH levels are suppressed, it suggests an ACTH-independent cause of Cushing's syndrome, while elevated ACTH levels suggest an ACTH-dependent cause. Further evaluation may include a CT scan of the chest, abdomen, and pelvis to identify potential ectopic sources, as well as an MRI of the pituitary gland [8]. Our patient had a high level of 24 h. urine free cortisol of 526.61 nmol/24 h (reference range: 30–145 nmol/24 h) and serum cortisol of 535.16 nmol/L(reference range: <50 nmol/L) after overnight 1 mg dexamethasone suppression test, but normal level of ACTH of 28.3 pg./ml (reference range: 7.2–63.1 ng/ml), this suggests the diagnosis of ACTH independent Cushing's syndrome. 4. Conclusion Large Pheochromocytoma patients can be asymptomatic and can present in association with other endocrine disorders. So proper evaluation is necessary to find out associated conditions and manage accordingly to prevent the possible outcomes. Patient consent Written, informed consent was obtained from the patient for the publication of the report. Ethical approval It is exempted at my institution. 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Cushing's syndrome (CS) is a rare disease with multiple somatic signs and a high prevalence of co-occurring depression. However, the characteristics of depression secondary to CS and the differences from major depression have not been described in detail. In this case, we report a 17-year-old girl with treatment-resistant depression with a series of atypical features and acute psychotic episodes, which is a rare condition secondary to CS. This case showed a more detailed profile of depression secondary to CS and highlighted the differences with major depression in clinical features, and it will improve insight into the differential diagnosis especially when the symptoms are not typical. Introduction Depression is a chronic medical problem with typical features, including sadness, decreased interest and cognitive impairments. In clinical practice, depression can occur in other medical conditions, especially endocrinopathies, making it a more complex problem and exhibiting a challenge in diagnosis, especially in first-contact patients or when the clinical presentations are atypical. It is generally accepted that patients who failed to respond to two or more adequate trials of first-line antidepressants for treatment of major depressive episode are considered to have treatment-resistant depression (TRD) (1). For patients with TRD, a throughout evaluation should be performed to investigate the underlying organic causes. Cushing's syndrome is a rare but serious endocrine disease due to chronic exposure to excess circulating glucocorticoids with multisystem effects (2). The etiology of CS can be divided into adrenocorticotropic hormone (ACTH)-dependent and ACTH-independent. It is characterized by a series of clinical features suggesting hypercortisolism, for example, metabolic abnormalities, hypertension and bone damages (3). A variety of neuropsychiatric symptoms, such as mood disturbance, cognitive impairment and psychosis, also occur in more than 70% CS patients (4). CS is life-threating if not timely diagnosed and treated, however, correct diagnosis can be delayed due to the wide range of phenotypes, especially when they are not classical (5). Previous studies suggested that major depression was the most common co-morbid complication in CS patients, with a prevalence of 50–81% (6). Haskett's study confirmed that 80% of subjects meet the criteria for major depression with melancholic features (7). As reported in most recent investigations, depression in CS was not qualitatively different from non-endocrine major depression and the similarity was even striking (3, 8). However, some studies showed different conclusions and suggested a high prevalence of atypical depressive features other than melancholic features in CS (9). TRD and anxious depression has also been reported in CS patients (10, 11). All of the above conclusions suggest the complexity of depression with CS, and no distinct features have been found pertaining to hypercortisolism (12, 13). Although the intensity of depression secondary to CS is severe, suicidal depression is still an unusual condition (14). Psychosis is a rare manifestation of CS, and the literature is limited. Only a few cases have been reported so far, especially combined with depression episode. In this case report, we presented a girl with CS, who experienced suicidal depression with a series of atypical features and acute psychotic symptoms, which was rarely reported in previous studies. Case description A 17-year-old girl with major depression for 3 years was involuntarily admitted for severe depressed mood with suicide attempts (neck cutting; tranquilizer overdose) and paranoid state in the last 2 weeks without any precipitating factors. She experienced depressed and irritable mood in the last 3 years, and her condition had not improved although several adequate trials of antidepressants were used with satisfactory compliance (sertraline 200 mg/d; escitalopram oxalate 20 mg/d). Over the 2 weeks prior to admission, her depression continued to worsen with increasing irritability, she committed several suicide attempts, and once stated that she was unsafe at home. On admission, her heart rate was 116 bpm with blood pressure 139/81 mmHg and normal temperature; physical examination showed a cushingoid and virilising appearance (central obesity, swollen and hirsute face with acne, purple striae on the abdomen and bruises on the arms). No other abnormal signs were noted. She seemed drowsy but arousable, and she walked slowly, with bent shoulders and an inclined head. Mental state examination was hard to continue because she was passive and reluctant to answer our questions. Venlafaxine 150 mg/d has been used for more than 3 months with poor effects at that time. Besides, weight gain (25 kg), irregular menstrual cycles and numbness of the hands and feet in the last half year were reported by her parents. Otherwise, No episodes of elevated mood and hyperactivity were found during the history taking. She does not have remarkable family history of serious physical or psychiatric illness; she was healthy, had an extroverted personality and had never used substances. Her premorbid social function and academic performance were good. Several clinical characteristics found during the following mental state examinations were listed as follows: • Prominent cognitive impairment without clouding of consciousness: Forgetfulness was frequently noted; she easily forgot important personal information such as her school and grade; she could not recall the suicide attempt committed recently and perfunctorily ascribed it to a casual event; and it was hard for her to recall her medical history (as it is for other depressive patients). The serial seven subtraction task could not be finished, and the interpretation of the proverb was superficial. Difficulty was found in attention maintenance; an effective conversation was hard to perform because she was mind-wandering (we needed to call her name to get her immediate attention) and often interrupted our conversations by introducing irrelevant topics or leaving without apparent reasons. • Decreased language function that did not match her educational background: The patient could not find the proper words to articulate her feelings; instead, many simple, obscure and contradictory words were used, which made her response seem perfunctory. For example, she responded with “I do not know,” “I forgot,” or kept silent in response to our questions, which made the conversations hard to perform. • Psychotic outbursts: Once she left the psychological therapy group, ranted about being persecuted and shook in fearfulness, stated “call the police” repeatedly, negative of explanations and comforts from others, but she cannot give any explanation about her behavior when calmed down. Sometimes she worried about being killed by the doctors but the worries were transient and fleeting. • Depressed mood and negative thoughts (self-blame, worthlessness, and hopelessness) that were not persistent and profound: During most of her hospitalization, the patient seemed confused and apathetic, with intermittent anxiety, but she could not clearly express what made her anxious. Her crying and sadness happened suddenly, without obvious reasons, and she even denied low mood sometimes and said she had come to the hospital for cardiac disease treatment (she did not have any cardiac disease). Her description of her depressed mood was uncertain when specifically questioned, and she rarely reported her depressed feeling spontaneously as other depressed patients would. She did not even have the desire to get rid of her “depression”. Her suicidal ideation was transient and impulsive, and she could not provide a comprehensive explanation for her suicide attempts, such as emptiness, worthlessness or guilt. She was impatient and restless when interacting with others or when a more in-depth conversation was performed. She seemed apathetic, gave little response to emotional support from others and did not care about relevant important issues, such as hospital discharge or future plans. Elevated mood and motor activity were not found during the admission period. • Social withdrawal and inappropriate behaviors: The patient often walked or stayed alone for long periods of time before speaking to other patients suddenly, which seemed improper or even odd in normal social interactions. During most hospitalization periods, lethargy and withdrawal were obvious. Diagnostic assessment and therapeutic interventions Basic laboratory tests reported abnormal results (Table 1), and the circulating cortisol level was far beyond the upper limit of normal, with a loss of circadian rhythm (Table 2); 24-h urinary free cortisol : >2897 nmol/24 h↑(69–345 nmol/24 h); serum ACTH (8 AM, 4 PM, 12 PM): 1.2 pg/ml, 1.3 pg/ml, <1 pg/ml (normal range: 1–46 pg/ml); low-dose dexamethasone suppression test (1 mg) (cortisol value): 1010.1 nmol/l (not suppressed; normal range: <50 nmol/L); high dose dexamethasone inhibition test (cortisol value): 879.0 nmol/l (not suppressed); OGTT and glycosylated hemoglobin; both normal. Other results used to rule out hyperaldosteronism and pheochromocytoma, such as the aldosterone/renin rate (ARR) and the vanillylmandelic acid, dopamine, norepinephrine and epinephrine levels, were reported to be within normal limits; ECG suggested sinus tachycardia; dual-energy X-ray bone density screening values were lower than the normal range; B-mode ultrasound showed a right adrenal tumor and fatty liver. The abdominal CT scan showed a tumor in her right adrenal gland. Brain MRI showed no abnormalities. Psychometric tests including HAMD (Hamilton depression scale), MADRS (Montgomery-Asberg Depression Rating Scale), WAIS (Wechsler Intelligence Scale) and MMSE (Mini-mental State Examination) were hard to perform due to her poor attention and non-cooperation presentation. Table 1 Table 1. Abnormal lab results for the patient. Table 2 Table 2. Circulating cortisol level. The patient had little response to adequate antidepressants in our hospital, including fluoxetine 20–60 mg/d and aripiprazole 5–30 mg/d combined with 3 sessions of MECT (modified electroconvulsive therapy), which was stopped because of her poor cognitive function and poor response. Her last diagnosis was right adrenal adenoma and non-ACTH-dependent Cushing's syndrome. The adrenal adenoma was excised through laparoscopic resection in a general hospital. Hydrocortisone, amlodipine besylate, potassium chloride, metoprolol and escitalopram were used for treatment. Escitalopram 10 mg/d has been used until 2 weeks after her discharge. At the follow-up visit about 1 month after the surgery, her depressive mood had significantly improved, with no self-injury behaviors or psychiatric symptoms found. The patient was calm but still reacted slowly, and cognitive impairment was still found at the last visit. Discussion Previous studies have reported a close association between CS and depression (15). However, suicidal depression with atypical features and acute psychosis have rarely been reported, and the characteristics of depression secondary to CS and the differences from major depression have not been described in detail. This case did not show a full-blown presentation of major depression according to the DSM-5. She presented with a series of features that were not typical as major depression, however, it should be emphasized that the atypical features were not identical to those noted in DSM5, especially regarding increased appetite and hypersomnia. The features suggesting difference from major depression were listed as follows: (a) depressed mood is not constant, it does not exist in most of the day; it is episodic without regular cyclicity, can happen or exacerbate suddenly; (b) the ability to describe anhedonia is poor, she can't report her feeling voluntarily like other patients with major depression, which might be partially related with the decreased language function; (c) depressive thoughts such as self-accusation and feelings of guilt, the classical symptoms of major depression, were rarely found; (d) more exaggerated cognitive impairment and decrease language function; € partial or little useful effect of SSRIs (selective serotonin reuptake inhibitors). The above characteristics were similar to those reported in Starkman's research (13, 16, 17), in which increasing irritability was also regarded as one of the important features for depression in CS. The literature about depression combined with psychosis episode in CS is rare. This patient showed acute episodes of persecutory delusion with disturbed behaviors; her psychotic symptoms occurred suddenly and were fragmentary, with poor sensitivity to antipsychotics; the content was not constant (she never referred to and even denied the unsafe feeling at home before admission), it changed with the environment and was not consistent with the mood state. However, we cannot reach an effective conclusion because the evidence was small; thus, these findings should be evaluated in combination with other clinical presentations. Conclusion Most reviews have concluded that mood disturbances in CS indicate “major depression”, but the detailed description of clinical features are lack, making clinicians uncertain about the presentation and confused about the diagnosis, especially when the somatic signs are indiscriminate. The clinical presentation in this case highlighted the fact that there is a wide range of phenotypes of depression in CS, for some CS patients, the depressive features are not highly consistent with the criteria of major depression regardless of the melancholic or atypical features in the DSM-5. Thus, a thorough and periodic evaluation is necessary to detect the underlying organic and psychosocial causes if the clinical symptoms are not typical (10). Data availability statement The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. Ethics statement Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article. Author contributions XY, SC, XJ, and XH were responsible for clinical care. XY did literature search and drafted the manuscript. 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Abstract The most common cause of Cushing syndrome (CS) is exposure to exogenous glucocorticoids. There is an increasing incidence of adulterated over-the-counter (OTC) supplements containing steroids. We present a case of Artri King (AK)-induced CS in a 40-year-old woman who presented with an intertrochanteric fracture of her right femur. Laboratory testing revealed suppressed cortisol and adrenocorticotropic hormone, which was consistent with suppression of the hypothalamic-pituitary-adrenal (HPA) axis. Following the cessation of the AK supplement, the patient’s HPA axis recovered, and the clinical manifestations of CS improved. This case emphasizes the need for better regulation of OTC supplements and the need for cautious use. Introduction Cushing syndrome (CS) is a condition that occurs because of high blood levels of glucocorticoids (GCs). These patients can present with a variety of systemic signs and symptoms, including truncal obesity, easy bruising of the skin, violaceous abdominal striae, resistant hypertension, dysglycemia, as well as osteoporosis. CS can occur because of adrenal etiologies such as adrenal adenoma, adrenal cancer, or adrenal hyperplasia or from an adrenocorticotropic hormone (ACTH)-producing pituitary adenoma or ectopic tumor. However, the most common cause of CS is the exogenous administration of GCs [1]. While exogenous GCs are often medically prescribed for the treatment of inflammatory conditions, some patients may be accidentally exposed to exogenous GCs from over-the-counter (OTC) supplements. We present a case of a young woman who developed exogenous CS and suffered a hip fracture as a result of taking an OTC supplement, Artri King (AK), adulterated with GCs. Case Presentation A 40-year-old obese woman presented to the hospital following a fall at home. She reported a snapping noise and sudden right hip pain while trying to stand up, and subsequently fell to the floor. She had noted right-sided hip pain for several days preceding her fall. She was evaluated in the emergency department where computed tomography (CT) imaging of the right lower extremity showed an intertrochanteric fracture of the right femur (Figure 1). The patient underwent open reduction and internal fixation of her right femur. The patient reported an unexplained weight gain of approximately 40 lbs in the preceding five months with a peak weight of 223 lbs (101 kg) and a body mass index (BMI) of 37 kg/m2. The patient denied taking any medications or supplements at the time of hospitalization. The endocrinology team was consulted to evaluate for causes of secondary osteoporosis in this young woman. Figure 1: A CT scan showing the right intertrochanteric fracture of the right femur (yellow arrows) Diagnostic assessment Her vital signs showed a blood pressure of 142/96 mmHg, heart rate of 68 beats per minute, temperature of 98.1°F (36.7°C), and 98% oxygenation on room air. Physical examination did not reveal abdominal striae or buffalo hump. She did have supraclavicular fat deposition and central obesity. No proximal muscle weakness was present. Laboratory tests were pertinent for decreased 25-hydroxy vitamin D, increased parathyroid hormone (PTH), and normal calcium (Table 1). These findings were consistent with secondary hyperparathyroidism due to vitamin D deficiency. Dual-energy X-ray absorptiometry (DEXA) scan revealed osteoporosis (Figures 2, 3 and Tables 2, 3). Further testing showed normal thyroid-stimulating hormone (TSH), estradiol, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), thus ruling out hyperthyroidism and primary ovarian insufficiency as possible causes of reduced bone mineral density (Table 1). Random cortisol was checked as hypercortisolism was suspected but it was found to be decreased along with decreased ACTH as well (Table 4). A cosyntropin stimulation test was performed, which showed decreased baseline cortisol with inappropriately decreased cortisol levels at 30 minutes and 60 minutes (Table 5). Given the discordance between the patient’s presentation and the lab results, assay interference was suspected, and further evaluation of the adrenal function was performed. Repeat labs using liquid chromatography-mass spectrometry (LCMS) assay again confirmed persistently low cortisol (Table 4). A 24-hour free urine cortisol was too low to quantify per assay despite the adequate volume. Further evaluation showed overall low adrenal steroids, including deoxycorticosterone, 17-hydroxyprogesterone, androstenedione, 11-deoxycortisol, pregnenolone, dehydroepiandrosterone sulfate, corticosterone, and progesterone. Lab test Patient's value Reference range 25-hydroxy vitamin D 12.8 ng/ml 30-100 ng/ml Parathyroid hormone (PTH) 86.2 pg/ml 10-66 pg/ml Serum calcium 9.5 ng/dl 8.8-10.5 mg/dl Thyroid-stimulating hormone (TSH) 2.49 mIU/L 0.36-3.74 mIU/L Estradiol 57.1 pg/ml 19.8-144.2 pg/ml Follicle-stimulating hormone (FSH) 5.4 mIU/ml 2.5-10.4 mIU/ml Luteinizing hormone (LH) 6 mIU/ml 1.9-12.5 mIU/ml Table 1: Patient's lab values on admission Figure 2: Dual-energy X-ray absorptiometry (DEXA) scan of the femoral neck showing osteopenia Figure 3: Dual-energy X-ray absorptiometry (DEXA) scan of the lumbar spine showing osteoporosis Region Area (cm2) Bone mineral content (g) Bone mineral density (g/cm2) T-score Peak reference Z-score Age-matched Femoral neck 4.76 3.53 0.742 -1.0 87 -0.7 91 Total 33.39 26.14 0.783 -1.3 83 -1.1 85 Table 2: Summary of dual-energy X-ray absorptiometry (DEXA) scan results of the femoral neck Region Area (cm2) Bone mineral content (g) Bone mineral density (g/cm2) T-score Peak reference Z-score Age-matched L1 10.79 7.56 0.701 -2.6 71 -2.4 73 L2 11.79 9.06 0.768 -2.4 75 -2.1 77 L3 12.70 9.98 0.786 -2.7 73 -2.4 75 L4 15.57 11.42 0.733 -3.0 69 -2.7 71 Total 50.86 38.03 0.748 -2.7 71 -2.5 73 Table 3: Summary of dual-energy X-ray absorptiometry (DEXA) scan results of the lumbar spine Lab test Patient's values while on Artri King Patient's values four weeks off of Artri King Reference range Random cortisol (routine assay) <0.64 μg/dL 7.3 μg/dL 5-25 μg/dL Adrenocorticotropic hormone (ACTH) 1.5 pg/ml 12 pg/ml 7.2-63.3 pg/ml Random cortisol (using liquid chromatography-mass spectrometry (LCMS) assay) 0.526 μg/dL N/A 5-25 μg/dL Table 4: Patient's cortisol and adrenocorticotropic hormone levels before and after stopping Artri King Cosyntropin stimulation test Patient value Reference range Baseline cortisol 1.64 μg/dL 5-25 μg/dL Cortisol after 30 minutes 1.33 μg/dL >18 μg/dL Cortisol after 60 minutes 6.48 μg/dL >18 μg/dL Table 5: Results of cosyntropin test while on Artri King Treatment She was started on teriparatide as well as vitamin D and calcium supplementation for the treatment of osteoporosis. Based on the aforementioned testing and the apparent symptoms of hypercortisolism, the patient was questioned again about the potential intake of steroids. She then recalled that she had been taking AK, an OTC supplement promoted for joint pain and arthritis. She reported that she had been taking two tablets of the supplement three times a day intermittently for the past three years. The patient neglected to bring it to the medical team’s attention before because she was under the impression that it was a multivitamin and did not have implications on her diagnosis. She was asked to stop the supplement and was educated about potential adrenal insufficiency symptoms and GC withdrawal. Outcome and follow up Repeat labs after four weeks off AK showed improved cortisol and ACTH levels indicating recovery of her hypothalamic-pituitary-adrenal (HPA) axis (Table 4). She lost 25 lbs in this time span with lifestyle modification. She continues teriparatide for osteoporosis, and monitoring of her bone mineral density is planned. Discussion This patient initially presented with a pathological fracture of her right femoral head. Given her young age, causes of secondary osteoporosis, including CS, were explored. The prevalence of osteoporosis in CS patients is 50% [2]. The effects of GC on bone health have been well studied. The major mechanism by which GC affects bone mineral density is by impairment of bone formation. GCs increase osteoblast and osteocyte apoptosis and decrease osteoblast function through their catabolic effects, which result in a dramatic decrease in bone formation rate. A prolonged lifespan of osteoclasts is observed with GC. A decrease in bone formation markers such as P1NP and osteocalcin has been observed in patients treated with GC [3]. Long-term GC use is associated with increased risk for fractures with a reported global prevalence of fractures of 30-50%. The risk for vertebral fractures is even higher, particularly in the thoracic and lumbar vertebrae. Interestingly, the risk for fracture with GC use peaks early in the course of treatment, often as early as three months into treatment, and declines rapidly after GC discontinuation [4]. An increased fracture risk has been described even with relatively low doses of GC (2.5-7.5 mg of prednisone or other equivalently dosed GC) and even with short-term use of under 30 days [5]. Our patient’s initial labs confirmed adrenal suppression despite our initial suspicion of CS, given her ongoing weight gain, central obesity, and osteoporosis. However, no obvious source of exogenous GC was identified. In most cases, the source of exogenous GC is easily identified through medication reconciliation; however, in our case, the patient was inadvertently exposed to steroids from an unregulated supplement, AK. The supplement’s ingredients were listed as glucosamine, chondroitin, collagen, vitamin C, curcumin, methylsulfonylmethane, nettle, and omega-3 fatty acids, with no mention of any steroid components. In a letter to the editor of the Internal Medicine magazine, several doctors published their concerns about a recent increase in CS cases associated with the use of AK and other similarly unregulated products [6]. Based on our literature search, three similar cases were published [7,8]. The reported cases developed CS after taking Artri King for several months, but none of them presented with a fracture. A warning by the U.S. Food & Drug Administration (FDA) was issued on April 20, 2022, indicating that FDA laboratory testing of this supplement confirmed the presence of undeclared drug ingredients, including dexamethasone, methocarbamol, and diclofenac. The FDA, however, was unable to confirm the exact amount of dexamethasone that these supplements contained [9]. Adverse events, including liver toxicity and death, were reported by the FDA. One study revealed that between 2007 and 2016, the FDA had issued more than 700 warnings about the sale of dietary supplements that contained unlisted and potentially dangerous ingredients. The majority of these supplements included those marketed for sexual enhancement, weight loss, or muscle building [10]. This case highlights the risks of undisclosed ingredients in OTC supplements. Conclusions In conclusion, we recommend that a thorough reconciliation of medication and supplements be obtained for all patients with CS. Supplements should be stopped and HPA axis testing should be repeated in patients with suspected exogenous GC exposure, even if steroids are not declared in the ingredients. It is also important to monitor such patients for adrenal insufficiency due to GC withdrawal and consider GC tapering if necessary. Our patient showed improvement in cortisol levels with no overt symptoms of adrenal insufficiency without the need for GC therapy. This case demonstrates the first case of AK-induced CS resulting in a pathological fracture. Given the increased use and availability of OTC supplements, this case highlights on the importance of detailed history-taking and the role of supplements in causing CS. This case also stresses the need for further education and counseling of our patients as well as tighter control on the manufacturing and sale of these supplements. References Lacroix A, Feelders RA, Stratakis CA, Nieman LK: Cushing's syndrome. Lancet. 2015, 386:913-27. 10.1016/S0140-6736(14)61375-1 Mancini T, Doga M, Mazziotti G, Giustina A: Cushing's syndrome and bone. Pituitary. 2004, 7:249-52. 10.1007/s11102-005-1051-2 Briot K, Roux 😄 Glucocorticoid-induced osteoporosis. RMD Open. 2015, 1:e000014. 10.1136/rmdopen-2014-000014 Canalis E, Mazziotti G, Giustina A, Bilezikian JP: Glucocorticoid-induced osteoporosis: pathophysiology and therapy. Osteoporos Int. 2007, 18:1319-28. 10.1007/s00198-007-0394-0 Waljee AK, Rogers MA, Lin P, et al.: Short term use of oral corticosteroids and related harms among adults in the United States: population based cohort study. BMJ. 2017, 357:j1415. 10.1136/bmj.j1415 Del Carpio-Orantes L, Quintín Barrat-Hernández A, Salas-González A: Iatrogenic Cushing syndrome due to fallacious herbal supplements. The case of Ortiga Ajo Rey and Artri King. Med Int Mex. 2021, 37:599-602. Patel R, Sherf S, Lai NB, Yu R: Exogenous Cushing syndrome caused by a "Herbal" supplement. AACE Clin Case Rep. 2022, 8:239-42. 10.1016/j.aace.2022.08.001 Mikhail N, Kurator K, Martey E, Gaitonde A, Cabrera C, Balingit P: Iatrogenic Cushing’s syndrome caused by adulteration of a health product with dexamethasone. JSM Clin Case Rep. 2022, 3: U.S. Food and Drug Administration. Public notification: Artri King contains hidden drug ingredients. (2022). Accessed: February 25, 2023: https://www.fda.gov/drugs/medication-health-fraud/public-notification-artri-king-contains-hidden-drug-ingredients. Tucker J, Fischer T, Upjohn L, Mazzera D, Kumar M: Unapproved pharmaceutical ingredients included in dietary supplements associated with US Food and Drug Administration warnings. JAMA Netw Open. 2018, 1:e183337. 10.1001/jamanetworkopen.2018.3337 From https://www.cureus.com/articles/153927-exogenous-cushing-syndrome-and-hip-fracture-due-to-over-the-counter-supplement-artri-king#!/

Nearly one-third of women with endogenous Cushing’s syndrome and normal bone mineral density have a low trabecular bone score, according to study data. “A large proportion of patients had degraded microarchitecture despite normal BMD,” Hiya Boro, DM, MD, MBBS, consultant in endocrinology, diabetes and metabolism at Aadhar Health Institute in India, and colleagues wrote. “The risk of fracture may be underestimated if BMD alone is measured. Hence, trabecular bone score should be added as a routine complementary tool in the assessment of bone health in patients with Cushing’s syndrome.” Data were derived from Boro H, et al. Clin Endocrinol. 2023;doi:10.1111/cen.14944. Researchers conducted a cross-sectional study at a single center in India from March 2018 to August 2019. The study included 40 women with overt endogenous Cushing’s syndrome and 40 healthy sex-matched controls. Seum and salivary cortisol and plasma adrenocorticotropic hormone (ACTH) were measured. Participants were considered ACTH independent if they had a level of less than 2.2 pmol/L. Areal BMD was measured at the lumbar spine, femoral neck, total hip and distal one-third of the nondominant distal radius. Low BMD for age was defined as a z score of less than –2. Trabecular bone score was measured at the lumbar spine. Fully degraded microarchitecture was defined as a trabecular bone score of 1.2 or lower and partial degradation was a trabecular bone score of 1.21 to 1.34. Of the 40 women with Cushing’s syndrome, 32 were ACTH-dependent and the other eight were ACTH independent. Of the independent group, seven had adrenal adenoma and one had adrenocortical carcinoma. Women with Cushing’s syndrome had lower BMD at the lumbar spine (0.812 g/cm2 vs. 0.97 g/cm2; P < .001), femoral neck (0.651 g/cm2 vs. 0.773 g/cm2; P < .001) and total hip (0.799 g/cm2 vs. 0.9 g/cm2; P < .001) than the control group. “No significant difference was noted in the distal radius BMD,” the researchers wrote. “This may be explained by the fact that cortisol excess predominantly affects trabecular rather than cortical bone.” Absolute trabecular bone score was lower in the Cushing’s syndrome group compared with controls (1.2 vs. 1.361; P < .001). Based on trabecular bone score, 42.5% of women with Cushing’s syndrome had fully degraded bone microarchitecture, 45% had partially degraded microarchitecture and 12.5% had normal microarchitecture. “In our study, 32.5% of patients had normal BMD with low trabecular bone score, thus highlighting the fact that patients may have normal BMD despite degraded microarchitecture,” the researchers wrote. “As such, assessment of BMD alone may underestimate the risk of fractures in patients with Cushing’s syndrome.” From https://www.healio.com/news/endocrinology/20230809/bmd-may-underestimate-bone-deterioration-for-women-with-endogenous-cushings-syndrome

Abstract Context Cushing’s disease (CD) is rare condition burdened by several systemic complications correlated to higher mortality rates. The primary goal of clinicians is to achieve remission, but it is unclear if treatment can also increase life expectancy. Aim To assess the prevalence of cortisol-related complications and mortality in a large cohort of CD patients attending a single referral centre. Materials and methods The clinical charts of CD patients attending a referral hospital between 2001 and 2021 were reviewed. Results 126 CD patients (median age at diagnosis 39 years) were included. At the last examination, 78/126 (61.9%) of the patients were in remission regardless of previous treatment strategies. Patients in remission showed a significant improvement in all the cardiovascular (CV) comorbidities (p < 0.05). The CV events were more frequent in older patients (p = 0.003), smokers and persistent CD groups (p < 0.05). Most of the thromboembolic (TE) and infective events occurred during active stages of the disease. The CV events were the most frequent cause of death. The standardized mortality ratio (SMR) resulted increased in persistent cases at the last follow-up (SMR 4.99, 95%CI [2.15; 9.83], p < 0.001) whilst it was not higher in those in remission (SMR 1.66, 95%CI [0.34; 4.85], p = 0.543) regardless of the timing or number of treatments carried out. A younger age at diagnosis (p = 0.005), a microadenoma (p = 0.002), and remission status at the last follow-up (p = 0.027) all increased survival. Furthermore, an elevated number of comorbidities, in particular arterial hypertension, increased mortality rates. Conclusions Patients with active CD presented a poor survival outcome. Remission restored the patients’ life expectancy regardless of the timing or the types of treatments used to achieve it. Persistent CD-related comorbidities remained major risk factors. Introduction Cushing’s disease (CD) is the most common cause of endogenous glucocorticoid excess due to uncontrolled adrenocorticotropic hormone (ACTH) secretion from a pituitary adenoma, for the most part a microadenoma [1]. A rare condition with an estimated incidence of 0.6—2.6 cases per million per year, it is burdened by high morbidity and mortality, for the most part linked to cardiovascular (CV) events. This is particularly true for active CD which is characterized by hypertension, diabetes mellitus, obesity and dyslipidaemia. The severity of the clinical picture seems to depend more on the duration of the disease rather than on the degree of cortisol elevation, although other confounding factors may affect the clinical phenotype [2]. Prompt diagnosis and resolution of hypercortisolemia are paramount to revert cortisol-related comorbidities and to improve life expectancy. Although new individualized medical treatment options for CD continue to evolve, transsphenoidal surgery (TSS) remains the first line treatment for potentially operable patients as it is the only treatment that seems to provide a rapid, long-lasting remission. Persistent and recurrent cases are nevertheless major concerns, since up to 50% of cases might require other treatment modalities to achieve disease control and those patients are once again exposed to cortisol excess that can negatively impact their survival [3]. An increased mortality has been noted in patients with active CD, while patients in remission show a markedly lower one. It is still unclear if mortality in these patients is higher than that in the general population. Some studies report a normal life expectancy [4,5,6,7,8] while others describe a persistently higher mortality [9,10,11]. One study reported finding a higher mortality as long as 10 years after remission, and only patients cured by a single TSS showed a normal life expectancy [12]. In view of these considerations, this study was designed to assess the prevalence of cortisol-related comorbidities/complications and mortality in a large group of CD patients attending a tertiary referral centre over the past 20 years. Other study aims were to evaluate the predictors of long-term outcomes and the impact of different treatments on life expectancy in CD patients. Materials and Methods One hundred twenty-six CD patients diagnosed between December 2001 and December 2021 were eligible for this monocentric, retrospective, observational study. Hypercortisolism was suspected on the basis of the patient’s clinical features and it was confirmed by appropriate hormonal testing [low dose dexamethasone suppression test (LDDST), 24-h urinary free cortisol (UFC) and late-night salivary cortisol (LNSC)] after excluding the possibility of exogenous glucocorticoid intake from any route [13]. UFC and LNSC were assessed at least in two different samples as recommended [14, 15]. The diagnosis of ACTH-dependent syndrome was confirmed on the strength of detectable ACTH levels (> 10 ng/L) and appropriate responses to a high dose dexamethasone suppression test (HDDST), corticotrophin releasing hormone (CRH) and/or desmopressin (DDAVP) tests [16]. All the patients underwent a pituitary magnetic resonance imaging (MRI); they also underwent bilateral inferior petrosal sinus sampling (BIPSS) when the results of hormonal tests were ambiguous. The pituitary origin of ACTH secretion was confirmed by biochemical remission after TSS, histology and/or post-operative hypoadrenalism. The results of clinical, biochemical and radiological tests as well as the treatments performed to control cortisol secretion (surgery, radiotherapy and/or medical therapy), any comorbidities (i.e., arterial hypertension, impaired glucose homeostasis, dyslipidaemia, overweight), any hormone deficiencies, any complications (i.e., CD-related events such as infective, CV and thromboembolic events) and any deaths recorded in the medical charts were collected. The disease severity at baseline was defined on the basis of the patient’s UFC values as mild (up to two-fold the upper limit of normal – ULN), moderate (between 2 and 5 times the ULN) or severe (over five-fold the ULN). Patient’s classification on the basis of disease activity are indicated in Supplementary material and methods sections. The presence of hypertension, glucose metabolism impairment, obesity, dyslipidaemia and hypopituitarism were defined as by specific Guidelines, Supplementary [19,20,21,22,23,24]. The current study was designed in accordance with the principles of the Declaration of Helsinki and approved by the Ethical Committee of the province of Padova (protocol code 236n/AO/22, date of approval 29 April 2022). The types of CD complications characterizing the patient were classified into three categories: CV, thromboembolic (TE), or infective (IN) events. Depending on the timing of its presentation, an event was classified as occurring: “prior” to diagnosis, “during” active CD or “after” CD remission. Events requiring hospitalization or iv antibiotic administration were registered as IN events. The causes of death were classified under the following headings: CV, infections, cancer, psychiatric complications leading to suicide, TE events or other (the last when none of the previous causes was applicable). Statistical analysis Categorical variables were reported as counts or percentages, and quantitative variables as median and interquartile ranges [IQR]. The comparisons between groups were performed with a Mann–Whitney sum rank test for independent quantitative variables; a Wilcoxon signed-rank test was run for dependent quantitative variables. As far as categorical variables were concerned, the McNemar test or a chi-square test were used for paired and unpaired data, respectively. A Cox regression analysis was performed to evaluate possible predictors for events and mortality based on the assumption of constant hazards over time. As time-dependent variables (e.g., achieving remission) did not meet this assumption, their survival analysis was performed using Kaplan–Meier analysis. Regarding complications, as there is usually a delay in CD diagnosis [25], Kaplan Meier curves for event free probability were calculated beginning 24 months prior to the diagnosis in order to include “prior” events possibly related to cortisol excess in our analysis. Vice versa, survival analysis for mortality was calculated beginning with the CD diagnosis date. Standardized mortality ratio (SMR) was calculated based on indirect age standardization in order to compare the observed deaths in our CD population with the expected number of deaths in the general population [26, 27]. A Fisher exact test was carried out to assess significant differences with respect to the general population and calculating the 95% confidence interval (95% CI) for SMR. The threshold for statistical significance was set at p-value < 0.05. Statistical analyses were performed with R: R-4.2.0 for Windows 10 (32/64 bit) released in April 2022 and R studio desktop version 4.2.0 (2022-04-22) for Windows 10 64 bit (R Foundation for Statistical Computing, Vienna, Austria, URL https://www.R-project.org/). An open-source calculator was also used to perform the Fisher exact test (http://www.openepi.com). Results Baseline The data of 167 CD patients attending the Centre between December 2001 and December 2021 were collected. The information regarding 41 patients were not included in the analysis because of insufficient follow-up data (i.e. patients referred for second opinion or for diagnostic workup or those with follow-up < 1 year from first line treatment). The remaining 126 patients presented a median age at diagnosis of 39 [31–50 years]; the female: male ratio was 3:1. The median follow-up was 130.5 months [72.5–201.5]. The patients’ clinical features at the time of diagnosis are outlined in Table 1. Table 1 The patients’ clinical features at the time of diagnosis Full size table The median UFC levels were 3.2 times the ULN [2–5.6]. Almost half of the cohort presented moderate cortisol excess (45/98, 45.9%), with lower proportions of the patients presenting mild (26/98, 26.5%) and severe disease (22/98, 27.6%). Most of the patients (91/113, 80.5%) had a microadenoma, including 29/91(31.9%) with negative imaging. The remaining 22 patients (19.5%) had a macroadenoma. Treatments Most of the patients underwent TSS as the first line treatment (113/126), only one patient underwent craniotomy. Eight patients received primary medical treatment, three received first-line radiotherapy and one underwent BA soon after diagnosis. Overall, 115 patients underwent pituitary surgery (one patient with a previous unsuccessful pituitary irradiation) and the remission rate was 60.9%. Relapses were observed in 46.7% of the cases after a median time of 56 [29–83] months. The second surgery proved less successful with respect to the first one; the remission rate was 43.2% (16/37); of these, 25% developed recurrence during the follow-up period. The median time to relapse was 66.5 [36–120] months. Only two patients underwent a third surgery; in both cases it was not curative (Supplementary Fig. 1) [27]. A 4th and a 5th TSS were performed in one of these for debulking purposes due to an aggressive pituitary lesion. Surgical remission was not affected by pre-treatment with cortisol-lowering medications neither before the first (p = 1.0) nor the second TSS (p = 0.88). Moreover, hormone control did not improve the surgical outcomes, although a tendency towards a higher remission rate was observed in those patients who showed good disease control before undergoing the second surgery (Supplementary Fig. 2) [27]. Overall, 34 patients received radiotherapy, either the conventional (18.5%) or the stereotactic type (81.5%). Remission was noted in 36.7% (11/30) of the patients with at least a 12-month post-radiotherapy follow-up. As expected, the longer the follow-up, the higher the remission rate; it was 41.67% (10/24) and 46.7% (7/15) at 5 and 10 years, respectively. Thirteen patients underwent BA and achieved complete remission. Excluding the patients with less than 12 months of follow-up, 4 out of 11 (36.4%) of the patients developed CTP-BADX/NS over a mean follow-up period of 110 [106 -329] months. Three patients out of the 11 were previously irradiated at pituitary level to control cortisol secretion. Four CD patients underwent unilateral adrenalectomy due to a dominant adrenal lesion consistent with chronic ACTH stimulation. Two (50%), harbouring unilateral adenomas larger than 5 cm, achieved remission after surgery; both cases were previously irradiated at the pituitary level. All but one of the 48 patients with persistent hypercortisolism at the last follow-up were on cortisol lowering medications. The untreated patient had a residual mild cortisol excess after TSS and medical therapy was discontinued because of multiple drug intolerance. At the last follow-up 28 patients were receiving monotherapy, and 19 were receiving combination treatment; 25 patients were receiving steroidogenesis inhibitors, 9 pituitary-target drugs and 13 a combination of the two compounds (Supplementary Table 1) [27]. Most of our patients achieved UFC normalization (complete control in 67.4%, partial control in 22.7%, uncontrolled in 10.9%). Data pertaining to a single patient with renal function impairment who presented falsely low UFC were not included in this analysis. When available, LNSC was restored in 14/41 cases (34.2%). No differences in the patients’ outcomes linked to the type of treatment prescribed (monotherapy vs combination treatment) or its target (adrenal vs pituitary) were found (data not shown). We also evaluated the extent of cortisol excess throughout the active phase of CD both for the patients presenting persistence at the last available follow-up (n = 48) and for those in remission after multiple therapies (i.e., late remission) (n = 33). As described in the material and methods section, disease activity for each year of active disease was defined on the basis of patients’ UFC levels. A minimum of three UFC measurements were registered every year and the median value was calculated. When data were missing, the patients were considered uncontrolled during that period. The results are reported in Supplementary Table 2 [27]; both the persistence and late remission groups showed UFC levels < 2xULN over more than 50% of the time span evaluated (58.8% and 73.6%, respectively). There was a progressive increase in the proportion of controlled patients over the observation period (Fig. 1). Fig. 1 Percentage of patients controlled during active CD Full size image Comorbidities The principal CD features at baseline and at the last follow-up examination were evaluated, (Supplementary Table 2). At time of diagnosis, no differences were observed as regards comorbidities between patients who achieved remission and those with persistent disease at baseline, (Supplementary Table 3). The patients in remission at the last examination showed a significant improvement in all the parameters considered; those with persistent CD did not (Table 2). Table 2 A comparison of Cushing’s disease features at baseline and at the last follow-up examination Full size table As far as hormone deficiencies were concerned, 42/126 (33.3%) of the patients developed at least one deficit due to previous treatments (Supplementary table 4) [27], including hypocortisolism due to BA. Neither the second surgery nor radiotherapy led to an increase in hypopituitarism (Supplementary Fig. 3) [27]. Complications and mortality As far as CD complications were concerned, 18.3% of the patients had a TE event, 17.5% presented an IN event and 7.1% presented a CV one. Most of the events occurred during an active phase of CD (Table 3). Other concomitant thrombotic risk factors were present in 10/19 (52.6%) of the patients experiencing TE events. TE events were related to surgery (pituitary, adrenal or others) in 5 cases, to post-traumatic fractures in 2, to prolonged immobilization in 2, and to a symptomatic SARS CoV2 infection in one case. IN events affected the respiratory system in 9 cases, the gastro-intestinal tract in 5 cases, the soft tissues in three cases, the central nervous system in 2 cases, the musculoskeletal system in 2 cases and the genitourinary tract in one case. Table 3 Thromboembolic, infective, and cardiovascular events and their timing (see materials and methods) Full size table Overall, 11 deaths were recorded during the follow-up period (130.5 [72.5–201.5] months). The causes of death were classified as: cardiovascular events (n = 4), infections (n = 2), cancer (n = 2), suicide (n = 1), thromboembolic events (n = 0), others (n = 2; a cerebral haemorrhage in one case and an unknown cause in the other). Cox regression was performed to evaluate the predictors of events (CV, IN, TE) and mortality (Fig. 2). The older patients presented an increased risk of mortality (HR 9.41, 95%CI [1.97; 44.90], p = 0.005), of CV events (HR 4.84, 95%CI [1.13; 20.75], p = 0.003) and of TE events (HR 2.41, 95%CI [1.02; 5.65], p = 0.04). Similarly, the presence of a macroadenoma at the time of the first MRI was associated with reduced survival (HR 9.29, 95%CI [2.30; 37.53], p = 0.002). Smoking was correlated to CV events (HR 5.33, 95%CI [1.33; 21.37], p = 0.02). Hypercortisolism severity at baseline did not affect the risk of complications or survival. No gender related differences were observed, although a tendency toward more CV events was noted in the males (p = 0.08). Fig. 2 Cox regression analysis for predictors of mortality and cardiovascular, infective or thromboembolic events; only significant results are shown. HR: Hazard ratio; CI: confidence interval; n: number, CV: cardiovascular; TE: thromboembolic. *p < 0.05 Full size image Kaplan Meier curves were plotted for complications (CV, IN and TE) and mortality in order to assess time-dependent variables (i.e., the number of comorbidities and the disease status at the last follow-up, the timing of remission and the disease activity in the patients with persistent CD at the last follow-up). We found that persistent disease and multiple comorbidities (at least 3) at the last follow-up were associated with increased CV events (p = 0.044 and p = 0.013, respectively) and mortality (p = 0.027 and p = 0.0057, respectively) (Fig. 3). The timing of remission did not influence the mortality or the risk of complications (data not shown). With regard to the patients with persistence, those presenting total/partial control for more than half of the follow-up period considered tended to have fewer CV and IN events (p = 0.078 and p = 0.074, respectively) (Fig. 3). Similarly, among patients with persistent cortisol excess the impaired circadian rhythm of secretion was associate to TE events and a trend to higher mortality (Supplementary Fig. 4). Sub-analysis of each comorbidity revealed that hypertension played a pivotal role during the follow-up period for CV complications (p = 0.011) and mortality (p = 0.0039). Similarly, dyslipidaemia was related to CV events (p = 0.046) and prediabetes/diabetes were associated to TE events (p = 0.035). A tendency toward increased mortality in the patients with impaired glucose homeostasis at the last follow-up was also noted (p = 0.052) (Data not shown). Fig. 3 Kaplan Meier curves for cardiovascular events based on: A) comorbidities at the last follow-up examination; disease status at the last follow-up examination; C) control during active disease for patients presenting persistence at the last follow-up. Kaplan Meier curves for survival plotting: D) comorbidities at the last follow-up examination; E) disease status at the last follow-up examination. Kaplan Meier curves for infective events based on: F) hormone control during active disease of patients presenting persistence at the last follow-up examination. FU: follow-up; CV: cardiovascular; IN: infective. *p < 0.05 Full size image The entire CD cohort presented an increased mortality, with a SMR of 3.22 (95%CI [1.70; 5.60], p = 0.002). Mortality was significantly higher in the patients with persistent disease (SMR 4.99, 95%CI [2.15; 9.83], p < 0.001), but it was similar to that of the general population in the patients in remission (SMR 1.66, 95%CI [0.34; 4.85], p = 0.543). The finding was independent of the timing or the modality used to achieve cortisol control; for the early remission group the SMR was 2.15 (95%CI [0.36; 7.11], p = 0.477) and for the late remission group it was 1.14 (95%CI [< 0.01; 5.62], p = 1.0). The length of remission period was 82 [38–139] for the early remission group vs 85 [21–136] for the late remission one. Discussion Study findings have confirmed that CD patients have a higher mortality and, as previously observed, the most common cause of death in these patients was, first of all, CV events and, secondly, infections [9]. Although there were no fatal TE events in our cohort, that type of complication was the most frequent one. As expected, the patients with persistent CD presented significantly increased mortality with respect to the general population. At the last follow-up examination the CD patients in remission had a mortality rate that was comparable to that of the general population regardless of the number of treatments needed to achieve remission. The finding is in contrast with the results of a multicentre study examining patients with more than 10 years of remission that reported finding a normal life expectancy only in the patients who achieved an early remission following a single TSS [12]. The better life expectancy in our series may be explained by an extensive use of cortisol-lowering medications in our centre during active phases of CD. There was moreover at least a partial control in the late remission group during over 70% of the years assessed; this might have had a positive effect on the overall survival rate (data not shown). Furthermore, our study considered relatively recent years when significant improvement in timely diagnosis and available medical therapies have been made [9]. Lastly, being monocentric, our study showed a homogenous management of comorbidities that by contrast, is in highly unlikely in a retrospective international study. Since cardiovascular and metabolic risk factors related to cortisol-excess are major determinant of mortality in CD, the latter point is of the outmost importance. Survival was positively influenced in our cohort by a younger age at diagnosis, the presence of a microadenoma at baseline [9] and a remission status at the last follow-up examination. As expected, an elevated number of comorbidities increased mortality, and as has been previously reported, arterial hypertension, in particular, reduced survival [28]. A tendency toward increased mortality was also noted in connection to impaired glucose homeostasis, but data on this topic are still controversial [8, 10, 12, 28, 29]. Cortisol excess atherosclerotic risk leading to CV events are closely liked. Beyond cortisol’s direct action on the tissues, this association is probably related to a clustering of several metabolic complications such as insulin resistance, arterial hypertension, dyslipidaemia and overweight commonly present in CD patients [30, 31]. Indeed, the patients presenting multiple comorbidities, especially arterial hypertension and dyslipidaemia, showed more CV complications. CV events were also more frequent in the patients with persistent hypercortisolism, and, as observed in general population in the elderly and in the smokers [32]. Older age at the time of diagnosis and dis-glycemia at the last follow-up examination were found to be related to TE events. It was instead impossible to identify predictors of infective complications. Although most TE and IN events occurred during active disease, remission did not significantly reduce these complications. The finding is in line with the data of a recent study focusing on a Swedish population reporting that CD patients present a higher risk of sepsis and thromboembolism even during long term remission [33]. Moreover, it is worthy of note that most of the TE events (52.6%) were accompanied by a concomitant risk factor such as recent surgery. These data highlight the importance of adequate prophylaxis in CD patients facing prothrombotic conditions such as those linked to a perioperative period [3, 34]. Disease severity at the baseline did not affect the patients’ complications or survival; the finding is not entirely surprising as the degree of cortisol excess does not necessarily correlate with the severity of the clinical picture [2]. The patients who achieved remission in our cohort showed an overall improvement in all the cortisol-related comorbidities. Hypertension was the most prevalent complication at the time of diagnosis, while overweight, which persisted in approximately 50% of the cases after remission, became by far the most frequent comorbidity. Glucose homeostasis alterations were the least prevalent at the time of diagnosis, although an underestimation is probable, as only fasting glycaemia or glycosylated haemoglobin were evaluated in most cases and provocative testing for hypercortisolism was not carried out [35]. With regards to demographic features, for the most part our patients were diagnosed during their third/fourth decade of life and they were prevalently female, in line with previous reports [36]. Most cases were due to a pituitary microadenoma (80% of the cases in our patients), including non-visible lesions on the MRI. As far as treatment was concerned, the remission rate after the first TSS was quite low with respect to what would be expected at a tertiary centre; the finding can be explained by the fact that many of the patients studied had been referred to our unit after undergoing unsuccessful pituitary surgery elsewhere. However, the assessment of surgical performance in various centres goes beyond the aim of the present study. As expected, a second TSS was less successful than the first one, but the rate of success found in our patients was in line with literature data [37]. Although the immediate remission rate after a second TSS was comparable to the long term outcome of radiotherapy, a quarter of the patients experienced a relapse just as they did after the first surgery [17]. Regarding the risk of developing hypopituitarism was concerned, no significant difference was found between the two approaches. These data have confirmed that both re-intervention and radiation treatment can be considered valid second-tier options, and a case by case approach should be adopted. Pre-operative medical treatment with cortisol-lowering medications did not improve the surgical outcomes, regardless of its effectiveness in controlling cortisol excess, in line with data by the European Registry on Cushing’s Syndrome (ERCUSYN) [38]. At the last follow-up examination, no differences in disease control were found when the treatment targets (pituitary vs adrenal) of the patients were compared. A higher control rate of hypercortisolism during active CD was found over time, possibly reflecting better drug dose titration and the widening landscape of available drugs with over two thirds of the patients presented completely controlled UFC at last examination. The fact that only one third of our patients achieved circadian rhythm restoration confirmed the previously reported difficulty in normalizing this parameter [39,40,41]. Interestingly, TE were more frequent when LNSC was uncontrolled and the same tendency was observed for survival, confirming the better outcome of patients with rhythm restoration [8]. Although only the last available value of LNSC was assessed, this finding might potentially turn the spotlight on the importance of LNSC normalization during medical treatment [42], but further studies are required to confirm these data. In line with previous reports, more than one third of the patients who underwent BA developed CTP-BADX/NS [18]. Although BA seems to immediately control hypercortisolism, this benefit should be carefully weighed against the risk of permanent adrenal insufficiency and CTP-BADX/NS. The patients received minimal doses of glucocorticoid replacement treatments following BA to avoid both over- and under treatment that might negatively impact survival [43], and this might explain why BA was not associated to increased mortality as observed in other series [44]. Unilateral adrenalectomy was performed in selected cases when a large adrenal nodule, probably provoked by chronic ACTH stimulation [45], was found. Interestingly, two patients who had previously undergone radiation treatment of the pituitary achieved disease remission after this surgery. The “transition” from pituitary to adrenal hypercortisolism after long standing ACTH-stimulation on adrenal nodules in CD patients has already been described by other investigators, and it may explain our findings in the patients studied [46]. The study’s retrospective single-centre nature represents its primary limitation. Its other important limitation, the relatively low number of cases and deaths examined, is of course linked to the condition’s rarity. Being a monocentric study does, on the other hand, have its advantages as it ensures that the treatment strategies, comorbidities evaluation and management are homogeneous. Furthermore, data on comorbidities, disease activity, type of cortisol lowering medications and comorbidities are available for most of our cohort. Besides, a potential protective effect of tailored medical therapy to reduce cortisol levels seems to reduce some complications and, to a less extent, overall mortality, especially when circadian cortisol secretion is restored. Further studies are still required to confirmed these latter findings. To conclude, active CD is characterized by increased morbidity and mortality, but disease remission seems to restore a normal life expectancy regardless of the timing and type of treatment used to achieve it. Thus, our aim as physicians is to pursue this goal by any means. Conversely, persistent cases seem to maintain an increase mortality, despite the use of effective cortisol lowering medications. Clearly persistent CD-related comorbidities require opportune monitoring and prompt management. Data availability Raw data are available from the corresponding author upon reasonable request. References Barbot M, Zilio M, Scaroni C (2020) Cushing’s syndrome: overview of clinical presentation, diagnostic tools and complications. Best Pract Res ClinEndocrinolMetab 34(2):101380. https://doi.org/10.1016/j.beem.2020.101380 Article CAS Google Scholar Guarnotta V, Amato MC, Pivonello R et al (2017) The degree of urinary hypercortisolism is not correlated with the severity of cushing’s syndrome. 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Author information Authors and Affiliations Endocrinology Unit, Department of Medicine-DIMED, University Hospital of Padova, Via Ospedale Civile, 105, 35128, Padua, Italy Alessandro Mondin, Filippo Ceccato, Giacomo Voltan, Pierluigi Mazzeo, Carla Scaroni & Mattia Barbot Neuroradiology Unit, University Hospital of Padova, Padua, Italy Renzo Manara Academic Neurosurgery, Department of Neurosciences, University of Padova, Padua, Italy Luca Denaro Contributions AM and MB wrote the main manuscript text, AM run statistics, AM prepared figures, GV and PM data collection and prepared tables, all authors were involved in patients’ management, CS and MB design the study, FC, CS and MB reviewed the manuscript. Corresponding author Correspondence to Mattia Barbot. Ethics declarations Competing interests Authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matters discussed in this manuscript. Ethical approval The current study was designed in accordance with the principles of the Declaration of Helsinki and approved by the Ethical Committee of the province of Padova (protocol code 236n/AO/22, date of approval 29 April 2022). Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 327 KB) From https://link.springer.com/article/10.1007/s11102-023-01343-2

YOU’RE INVITED! A4M/Metabolic Medical Institute (MMI) Webinar on Updates on Treating Hypothyroidism Dr. Theodore Friedman (The Wiz) will giving a webinar on Updates on Treating Hypothyroidism Topics to be discussed include: New articles showing patients prefer desiccated thyroid Moving away from a TSH-centered approach New thyroid hormone preparations, including Adthyza •Does biotin affect thyroid tests? •Hypothyroidism diet? What is the difference between desiccated thyroid and synthetic thyroid hormones? Is rT3 important? Wednesday • August 2, 2023 • 4 PM PDT register at https://us02web.zoom.us/webinar/register/WN_kRyqZFlrSgKj54CuK7OQqQ Slides will be available on the day of the talk here. There will be plenty of time for questions using the chat button. For more information, email us at mail@goodhormonehealth.com

The most common procedure to remove pituitary tumors is transsphenoidal adenomectomy. It allows the removal of the tumor with minimal damage to the surrounding structures. The surgical team accesses the pituitary gland through the sphenoid sinus — a hollow space behind the nasal passages and below the pituitary gland. If performed in specialized centers and by an experienced pituitary surgeon, this type of surgery is reported to result in an overall cure rate, or full remission, of Cushing’s disease for 80% to 90% of patients. A higher success rate is seen with smaller tumors. However, reported remission rates vary considerably, mainly due to differences in the criteria used to define disease remission. In some cases, a second transsphenoidal adenomectomy is required to fully remove tumor tissue; in others, the initial surgical procedure is paired with a second form of treatment, such as radiation therapy or certain medications. Given the complexity of the procedure, the guidelines recommend patients undergo surgery in specialized Pituitary Tumor Centers of Excellence. Patients also are advised to have the surgery performed by an experienced pituitary neurosurgeon. Follow-up for all patients should be conducted by a multidisciplinary team, including a pituitary endocrinologist. Lifelong monitoring for disease recurrence is required.

Abstract Purpose. Few related factors of low bone mass in Cushing’s disease (CD) have been identified so far, and relevant sufficient powered studies in CD patients are rare. On account of the scarcity of data, we performed a well-powered study to identify related factors associated with low bone mass in young CD patients. Methods. This retrospective study included 153 CD patients (33 males and 120 females, under the age of 50 for men and premenopausal women). Bone mineral density (BMD) of the left hip and lumbar spine was measured by dual energy X-ray absorptiometry (DEXA). In this study, low bone mass was defined when the Z score was −2.0 or lower. Results. Among those CD patients, low bone mass occurred in 74 patients (48.37%). Compared to patients with normal BMD, those patients with low bone mass had a higher level of serum cortisol at midnight (22.31 (17.95-29.62) vs. 17.80 (13.75-22.77), ), testosterone in women (2.10 (1.33–2.89) vs. 1.54 (0.97–2.05), ), higher portion of male (32.43% vs. 11.54%, ) as well as hypertension (76.12% vs. 51.67%, ), and lower IGF-1 index (0.59 (0.43–0.76) vs. 0.79 (0.60–1.02), ). The Z score was positively associated with the IGF-1 index in both the lumbar spine (r = 0.35153, ) and the femoral neck (r = 0.24418, ). The Z score in the femoral neck was negatively associated with osteocalcin (r = −0.22744, ). Compared to the lowest tertile of the IGF-1 index (<0.5563), the patients with the highest tertile of the IGF-1 index (≥0.7993) had a lower prevalence of low bone mass (95% CI 0.02 (0.001–0.50), ), even after adjusting for confounders such as age, gender, duration, BMI, hypertension, serum cortisol at midnight, PTH, and osteocalcin. Conclusions. The higher IGF-1 index was independently associated with lower prevalence of low bone mass in young CD patients, and IGF-1 might play an important role in the pathogenesis of CD-caused low bone mass. 1. Introduction Cushing’s disease (CD), caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, is a rare disease with approximately 1.2 to 2.4 new cases per million people each year [1]. Osteoporosis has been recognized as a serious consequence of endogenous hypercortisolism since the first description in 1932 [2]. The prevalence of osteoporosis is around 38–50%, and the rate of atraumatic compression fractures is 15.8% in CD patients [3]. After cortisol normalization and appropriate treatment, recovery of the bone impairment occurs slowly (6–9 years) and partially [4, 5]. Hypercortisolemia impairs bone quality through multiple mechanisms [6]. Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) play a crucial role in bone growth and development [7]. IGF-1 is considered essential for the longitudinal growth of bone, skeletal maturity, and bone mass acquisition not only during growth but also in the maintenance of bone in adults [8]. Previous research studies revealed that low serum IGF-1 levels were associated with a 40% increased risk of fractures [9, 10], and serum IGF-1 levels could be clinically useful for evaluating the risk of spinal fractures [11]. In Marl Hotta’s research, extremely low or no response of plasma GH to recombinant human growth hormone (hGRH) injection was noted in CD patients. This result suggested that the diminished hGRH-induced GH secretion in patients with Cushing’s syndrome might be caused by the prolonged period of hypercortisolemia [12]. Other surveys indicated that glucocorticoids, suppressing GH–IGF-1 and the hypothalamic-pituitary-gonadal axes, lead to decreased number and dysfunction of osteoblast [13]. However, the exact mechanism is still unclear, and few risk factors for osteoporosis in CD have been identified so far. Until now, relevant and sufficiently powered studies in CD patients have been rare [14, 15]. Early recognition of the changes in bone mass in CD patients contributes to early diagnosis of bone mass loss and prompt treatment, which could help minimize the incidence of adverse events such as fractures. On account of the scarcity of data and pressing open questions concerning risk evaluation and management of osteoporosis, we performed a well-powered study to identify the related factors associated with low bone mass in young CD patients at the time of diagnosis. 2. Materials and Methods 2.1. Subjects This retrospective study enrolled 153 CD patients (33 males and 120 females) from the Department of Endocrinology and Metabolism of Huashan Hospital between January 2010 and February 2021. All subjects were evaluated by the same group of endocrinologists for detailed clinical evaluation. This study, which was in complete adherence to the Declaration of Helsinki, was approved by the Human Investigation Ethics Committee at Huashan Hospital, Fudan University (No. 2017M011). We collected data on demographic characteristics, laboratory tests, and bone mineral density. Inclusion criteria included the following: (1) willingness to participate in the study; (2) premenopausal women ≥18 years old, men ≥18 years old but younger than 50 years old, and young women (<50 years old) with menstrual abnormalities who were associated with CD after excluding menstrual abnormalities caused by other causes; (3) diagnosis of CD according to the updated diagnostic criteria [16]; and (4) pathological confirmation after transsphenoidal surgery (positive immunochemistry staining with ACTH). Exclusion criteria included Cushing’s syndrome other than pituitary origin. 2.2. Clinical and Biochemical Methods IGF-1 was measured using the Immulite 2000 enzyme-labeled chemiluminescent assay (Siemens Healthcare Diagnostic, Surrey, UK). Other endocrine hormones, including cortisol (F), 24-hour urinary free cortisol (24hUFC), adrenocorticotropic hormone (ACTH), prolactin (PRL), luteinizing hormone (LH), follicle stimulating hormone (FSH), estrogen (E2), progesterone (P), testosterone (T), thyroid stimulating hormone (TSH), and free thyroxine (FT4), were carried out by the chemiluminescence assay (Advia Centaur CP). Intra-assay and interassay coefficients of variation were less than 8 and 10%, respectively, for the estimation of all hormones. Bone metabolism markers included osteocalcin (OC), type I procollagen amino-terminal peptide (P1NP), parathyroid hormone (PTH), and 25-hydroxyvitamin D (25(OH)VD), measured in a Roche Cobas e411 analyzer using immunometric assays (Roche Diagnostics, Indianapolis, IN, USA). The IGF-1 index was defined as the ratio of the measured value to the respective upper limit of the reference range for age and sex. Body mass index (BMI) was calculated using the following formula: weight (kg)/height2 (m2). The bone mineral density (BMD) measuring instrument was Discovery type W dual energy X-ray absorptiometry from the American HOLOGIC company. Quality control tests were conducted every working day. Before examination, the date of birth, height, weight, and menopause date of the examiner were accurately recorded, and then BMD (g/cm2) of the left hip and lumbar spine were measured by DEXA. Z value was used for premenopausal women and men younger than 50 years old, and Z-value = (measured value − mean bone mineral density of peers)/standard deviation of BMD of peers [17, 18]. In this study, low bone mass was defined as a Z-value of −2.0 or lower. 2.3. Statistical Analysis The baseline characteristics were compared between CD patients with and without low bone mass by using the Student’s t-test for continuous variables and the χ2 test for category variables. Bone turnover markers, alanine aminotransferase (ALT), triglyceride (TG), IGF-1 index, thyroid stimulating hormone (TSH), free triiodothyronine (FT3), free thyroxine (FT4), testosterone (T), 24 hours of urine cortisol (24 h UFC), and serum cortisol at 8 a.m. (F8 am) and at midnight (F24 pm) were not in normal distribution, so variables mentioned above were Log10-transformed, which could be used as continuous variables during statistical analysis. Participants were categorized into three groups according to tertiles of the IGF-1 index: <0.5986, 0.5986–0.8380, and >0.8380. The linear trend across IGF-1 index tertiles was tested using linear regression analysis for continuous variables and the Cochran–Armitage test for categorical variables. We used a multivariate logistic regression model to identify related factors that are independently associated with the risk of low bone mass. Variables included in the multivariate logistic regression model were selected based on the Spearman rank correlation analysis and established traditional low bone mass risk factors as priors. The results were presented as odds ratios (OR) and the corresponding 95% confidence intervals (CI). Significance tests were two-tailed, with value <0.05 considered statistically significant for all analyses. Statistical analysis was performed using SAS version 9.3 (SAS Institute Inc, Cary, NC, USA). 3. Results 3.1. The Prevalence of Low Bone Mass in Young Cushing’s Disease Patients From the inpatient system of Huashan hospital, a total of 153 CD patients under the age of 50 for men and premenopausal women (some with menstrual abnormalities were associated with CD) were included, aged from 13 to 49 years, with an average age of 34.25 ± 8.39 years. There were 33 males (21.57%) and 120 females (78.43%). These CD patients included newly diagnosed CD, recurrences of CD, and CD without remission after treatment. There were no differences in the prevalence of different statuses of CD between the two groups (Table 1). Table 1 Clinical and biochemical preoperative characteristics of young Cushing’s disease patients according to status of bone mineral density at diagnosis. Among these CD patients, low bone mass occurred in 74 patients (48.37%), including 24 men and 50 women. The prevalence of low bone mass was 41.67% and 72.73% in female and male CD patients, respectively, and 42 (56.76%) patients suffered from low bone mass in the lumbar spine only, while 10 (13.51%) patients had low bone mass in the femoral neck only, and 22 (29.73%) patients had low bone mass in both parts. In female patients with low bone mass, 27 (54%) had low bone mass in the lumbar region only, 9 (18%) in the femoral neck only, and 14 (28%) had low bone mass in both parts. For male patients with low bone mass, 16 (66.67%) patients had low bone mass only in the lumbar region, and the rest (8, 33.33%) had low bone mass in both parts. Ten patients had a history of fragility fractures (6 ribs, 3 vertebrae, 1 femoral neck, and ribs), and all of them achieved low bone mass in BMD. 3.2. Baseline Characteristics of Cushing’s Disease Patients with and without Low Bone Mass These CD patients were divided into two groups with and without low bone mass (Table 1). Compared to patients without low bone mass, those low bone mass patients had a higher level of diastolic blood pressure (DBP) (97.07 ± 13.69 vs. 89.76 ± 13.43, ), serum creatinine (66.15 ± 24.33 vs. 55.90 ± 13.35, ), uric acid (0.36 ± 0.10 vs. 0.32 ± 0.10, ), cholesterol (5.57 ± 1.30 vs. 5.06 ± 1.47, ), testosterone in women (2.10 (1.33–2.89) vs. 1.54 (0.97–2.05), ), F24 pm (22.31 (17.95–29.62) vs. 17.80 (13.75–22.77), ), and higher portion of male (32.43% vs. 11.54%, ), as well as hypertension (76.12% vs. 51.67%, ). The low bone mass group had a lower IGF-1 index (0.59 (0.43–0.76) vs. 0.79 (0.60–1.02), ) and FT3 level (3.54 (3.16–4.04) vs. 3.98 (3.47–4.45), ) than those without low bone mass. CD patients without low bone mass were more likely to have serum IGF-1 above the upper limit of the normal reference range (ULN) with age-adjusted (18, 26.87% vs. 3, 4.84%, ). No differences of bone turnover makers were found between the two groups. 3.3. Association between Baseline Characteristics and BMD Spearman’s rank correlation analysis was used to explore the related factors of low bone mass in young CD patients (Table 2). The results indicated that the Z score in the lumbar spine was positively associated with age at diagnosis (r = 0.18801, ), IGF-1 index (r = 0.35153, ), FT3 level (r = 0.24117, ), estradiol in women (r = 0.2361, ), and occurrence of normal menstruation in females (r = 0.2267, ). Meanwhile, SBP (r = −0.21575, ), DBP (r = −0.32538, ), ALT (r = −0.17477, ), serum creatinine (r = −0.36072, ), cholesterol (r = −0.20205, ), testosterone in women (r = −0.2700, ), F8 am (r = −0.18998, ), and serum cortisol at midnight (r = −0.27273, ) were negatively associated with the Z-score in the lumbar spine. The results also illustrated that the Z-score in the femoral neck was positively associated with BMI (r = 0.33926, ), IGF-1 index (r = 0.24418, ), FT3 level (r = 0.20487, ), and occurrence of normal menstruation in females (r = 0.2393, ). Serum creatinine (r = −0.1932, ), osteocalcin (r = −0.22744, ), and testosterone in women (r = −0.2363, ) were negatively associated with the Z-score in the femoral neck. Table 2 Spearman rank correlation of BMD and various variables in Cushing’s disease patients. 3.4. IGF-1 Index and Low Bone Mass Participants were categorized into the following three groups according to tertiles of the preoperative IGF-1 index: <0.5986 (tertiles 1), 0.5986–0.8380 (tertiles 2), and >0.8380 (tertiles 3). With the IGF-1 index increasing, the level of PTH decreased (54.85 (38.35–66.2), 38.9 (26.6–66.9), 36 (25.5–47.05), and ), while other bone metabolism makers, including PINP, osteocalcin, and 25 (OH) VD, showed no differences among the three groups (Figures 1(a)–1(d)). With the increase in the IGF-1 index level, the Z-score of both vertebra lumbalis (tertiles 1: −2.4 (−3.3∼−1.5); tertiles 2: −1.9 (−2.3∼−1.0); tertiles 3: −1.15 (−1.9∼−0.4), ) and the neck of femur (tertiles 1: −1.7 (−2.3∼−0.95); tertiles 2: −1.2 (−1.9∼−0.5); tertiles 3: −1.0 (−1.5∼−0.5), ) increased gradually (Figures 2(a) and 2(b)). Meanwhile, prevalence of low bone mass decreased (68.29%, 53.33%, 23.81%, ) (Figure 3(a)) both in the vertebra lumbalis (63.41%, 48.89%, 16.67%, ) and the neck of femur (32.5%, 11.11%, 11.19%, ), with the increasing of the IGF-1 index level (Figures 3(b) and 3(c)). (d) (a) (b) (c) (d) (a) (b) (c) (d) (a) (b) (c) (d) Figure 1 Bone turnover makers in three groups according to tertiles of the preoperative IGF-1 index. Tertiles 1: <0.5986, tertiles 2: 0.5986–0.8380, and tertiles 3 >0.8380. a for PINP; b for osteocalcin; c for PTH; d for VD-OH25. (a) p for trend = 0.2601. (b) p for trend = 0.1310. (c) p for trend = 0.008. (d) p for trend = 0.7956. (b) (a) (b) (a) (b) (a) (b) Figure 2 Z-score of both the neck of femur and the vertebra lumbalis in three tertiles of the IGF-1 index. a for the neck of femur; b for the vertebra lumbalis. Tertiles 1: <0.5986, tertiles 2: 0.5986–0.8380, and tertiles 3 >0.8380. (a) p for trend = 0.0148. (b) p for trend < 0.0001. (c) (a) (b) (c) (a) (b) (c) (a) (b) (c) Figure 3 Prevalence of low bone mass according to tertiles of the preoperative IGF-1 index. With increment of the IGF-1 index level, prevalence of low bone mass decreased, both in the vertebra lumbalis and neck of femur. Tertiles 1: <0.5986, tertiles 2: 0.5986–0.8380, and tertiles 3 >0.8380. (a) p for trend = 0.0002. (b) p for trend = 0.0169. (c) p for trend < 0.0001. In the logistic regression analysis of the related factors of low bone mass, most of the potentially relevant factors were put into this model; only the IGF-1 index was still significantly negatively associated with the prevalence of low bone mass after adjusting for covariables. The results indicated that compared to the patients in the lowest tertile of the IGF-1 index (<0.5563), those with the highest tertile of the IGF-1 index (≥0.7993) had a lower prevalence of low bone mass (95% CI 0.16 (0.06–0.41), ). After adjusting for age, gender, and BMI, the patients in the highest tertile of the IGF-1 index still conferred a lower prevalence of low bone mass (95% CI 0.15 (0.06–0.42), ). The association between the IGF-1 index and low bone mass still existed (95% CI 0.02 (0.001–0.5), ) even after adjusting for age, gender, CD duration, BMI, hypertension, dyslipidemia, diabetes, ALT, Scr, FT3, F24 pm, PTH, and osteocalcin (Table 3). In comparison to the reference population, the participants in the middle tertile of the IGF-1 index (0.5563–0.7993) had no different risk of low bone mass. Table 3 Association between the preoperative IGF-1 index and the risk of low bone mass. 4. Discussion Our results revealed that low bone mass occurred in around half of young CD patients, affecting more males than females, and mostly in the lumbar spine. The CD patients in our study had a high prevalence (48.37%) of low bone mass at the baseline. This was in accordance with the findings of previous research, and the reported prevalence of osteoporosis due to excess endogenous cortisol ranges from 22% to 59% [19–25]. In this study, CD patients’ lumbar vertebrae were more severely affected than the neck of the femur. It is reported that lumbar vertebrae, containing more trabecular bone than femur neck, were more vulnerable to endogenous cortisol [26]. Our results also indicated that men were more prone to low bone mass than women in CD, which was in accordance with several other studies [23, 27, 28]; possibly, the deleterious effect of cortisol excess on BMD might overrule the protective effects of sex hormones, and men were more often hypogonadal compared with women in CD patients. In our study, patients with low bone mass had a significantly higher level of F24 pm. Both cortisol levels in the morning and at midnight, were negatively associated with the Z-score of BMD in the lumbar spine at diagnosis. But these results were not seen in the femoral neck at diagnosis. This further indicated that lumbar vertebrae were more vulnerable to endogenous cortisol. BMI was considered to be associated with bone mass [29]. In our study, higher BMI was associated with higher BMD at diagnosis in the femur neck but not in the lumbar vertebrae, consistent with other studies [30]. Interestingly, besides the above known related factors, we also found that a higher level of the IGF-1 index was strongly associated with a lower prevalence of low bone mass, both in the vertebra lumbalis and the neck of the femur, independently of age, gender, duration, BMI, hypertension, dyslipidemia, diabetes, level of ALT, creatinine, FT3, and F24 pm. The IGF-1 index was also positively associated with the BMD Z-score, both in the lumbar spine and the femoral neck. So far, there have been few studies concerning the association between IGF-1 and low bone mass in Cushing’s disease patients. As we know, GH [31, 32] and IGF-1 [33] have been demonstrated to increase both bone formation (e.g., collagen synthesis) and bone resorption. However, in CD patients, glucocorticoids resulted in decreased number and dysfunction of osteoblasts by inhibiting GH-IGF-1 axes [34, 35]. In vitro studies suggested that at high concentrations of glucocorticoids, a decreased release of GHRH had been reported [36–38]; therefore, GH-IGF-1 axes were inhibited. IGF-1 possessed anabolic mitogenic actions in osteoblasts while reducing the anabolic actions of TGF-β [39]. The decrease in IGF-1 might be a risk factor for low bone mass in CD patients. In vitro studies had also indicated that the suppressive effects of glucocorticoids on osteoblast function can be partially reversed by GH or IGF treatment [8]. In recent years, some studies have also shown that patients with untreated Cushing’s disease may have elevated IGF-1, and mildly elevated IGF-1 in Cushing’s disease does not imply pathological growth hormone excess. Higher IGF-1 levels could predict better outcomes in CD [40, 41]. Possible mechanisms were not clear, which might involve changes in IGF binding proteins (IGFBPs), interference in IGFBP fragments, IGF-1 synthesis or clearance, and/or the effects of hyperinsulinism induced by excess glucocorticoids. In our study, the results also showed that IGF-1 was an independent protective factor for low bone mass in CD patients. Our study was one of the few well-powered research studies on the association of IGF-1 levels with low bone mass in young CD patients. These represented important strengths of our study, especially given the rarity of CD. The main limitation of this study was its retrospective nature. This could not prove causality. A prospective study should be conducted to explore the causality between IGF-1 and osteoporosis in CD patients. In addition, this study lacked morphometric data for spinal fractures in all patients, which may underestimate the incidence of fractures and osteoporosis. However, our study indicated that a lower IGF-1 index level was significantly associated with low bone mass in young CD patients, which might provide a new aspect to understand the possible risk factors and mechanism of osteoporosis in CD patients. In conclusion, our study found that a higher IGF-1 index was independently and significantly associated with decreased prevalence of low bone mass in young CD patients, drawing attention to the role of IGF-1 in the pathogenesis of CD-caused low bone mass and may support the exploration of this pathway in therapeutic agent development in antiosteoporosis in CD. Data Availability The data used to support the findings of the study are available on request from the authors. Additional Points Through a retrospective study of a large sample of Cushing’s disease (CD) patients from a single center, we found that a higher IGF-1 index was independently associated with a lower prevalence of low bone mass in young CD patients and IGF-1 might play an important role in the pathogenesis of CD-caused low bone mass. Disclosure Wanwan Sun and Quanya Sun were the co-first authors. Conflicts of Interest The authors declare that they have no conflicts of interest. Authors’ Contributions Wanwan Sun analyzed the data and wrote the manuscript. Quanya Sun collected the data. Hongying Ye and Shuo Zhang conducted the study design and quality control. All authors read and approved the final manuscript. Wanwan Sun and Quanya Sun contributed equally to this work. Acknowledgments The present study was supported by grants from the initial funding of the Huashan Hospital (2021QD023). 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Abstract Objectives To assess the diagnostic performance of high-resolution contrast-enhanced MRI (hrMRI) with three-dimensional (3D) fast spin echo (FSE) sequence by comparison with conventional contrast-enhanced MRI (cMRI) and dynamic contrast-enhanced MRI (dMRI) with 2D FSE sequence for identifying pituitary microadenomas. Methods This single-institutional retrospective study included 69 consecutive patients with Cushing’s syndrome who underwent preoperative pituitary MRI, including cMRI, dMRI, and hrMRI, between January 2016 to December 2020. Reference standards were established by using all available imaging, clinical, surgical, and pathological resources. The diagnostic performance of cMRI, dMRI, and hrMRI for identifying pituitary microadenomas was independently evaluated by two experienced neuroradiologists. The area under the receiver operating characteristics curves (AUCs) were compared between protocols for each reader by using the DeLong test to assess the diagnostic performance for identifying pituitary microadenomas. The inter-observer agreement was assessed by using the κ analysis. Results The diagnostic performance of hrMRI (AUC, 0.95–0.97) was higher than cMRI (AUC, 0.74–0.75; p ≤ .002) and dMRI (AUC, 0.59–0.68; p ≤ .001) for identifying pituitary microadenomas. The sensitivity and specificity of hrMRI were 90–93% and 100%, respectively. There were 78% (18/23) to 82% (14/17) of the patients, who were misdiagnosed on cMRI and dMRI and correctly diagnosed on hrMRI. The inter-observer agreement for identifying pituitary microadenomas was moderate on cMRI (κ = 0.50), moderate on dMRI (κ = 0.57), and almost perfect on hrMRI (κ = 0.91), respectively. Conclusions The hrMRI showed higher diagnostic performance than cMRI and dMRI for identifying pituitary microadenomas in patients with Cushing’s syndrome. Key Points • The diagnostic performance of hrMRI was higher than cMRI and dMRI for identifying pituitary microadenomas in Cushing’s syndrome. • About 80% of patients, who were misdiagnosed on cMRI and dMRI, were correctly diagnosed on hrMRI. • The inter-observer agreement for identifying pituitary microadenomas was almost perfect on hrMRI. Introduction Cushing’s syndrome, caused by excessive exposure to glucocorticoids, is associated with considerable morbidity and increased mortality [1]. Cushing’s syndrome has diverse manifestations, including central obesity, moon facies, purple striae, and hypertension [2]. Cushing’s disease, due to adrenocorticotropic hormone (ACTH) hypersecretion from pituitary adenomas, is the most common etiology of ACTH-dependent Cushing’s syndrome [1, 2]. According to the Endocrine Society Clinical Practice Guideline, transsphenoidal surgery is the first-line treatment for Cushing’s disease [3]. The identification of pituitary adenomas on preoperative MRI can significantly increase the postoperative remission rate from 50 to 98% [4]. Therefore, it is critical to identify pituitary adenomas on MRI before surgery. However, there are considerable challenges in identifying ACTH-secreting pituitary adenomas. This is because about 90% of the tumors are microadenomas (less than 10 mm in size) and the median diameter at surgery is about 5 mm [5, 6]. Conventional contrast-enhanced MRI (cMRI) using a two-dimensional (2D) fast spin echo (FSE) sequence has been routinely used to acquire images with 2- to 3-mm slice thickness, but some microadenomas are difficult to be identified on cMRI, resulting in false negatives reported in up to 50% of patients with Cushing’s disease [7]. Dynamic contrast-enhanced MRI (dMRI) increases the sensitivity of identifying pituitary adenomas to 66% [8], but it also increases false positives at the same time [9, 10]. The 3D spoiled gradient recalled (SPGR) sequence has been introduced in high-resolution contrast-enhanced MRI (hrMRI) to acquire images with 1- to 1.2-mm slice thickness. It is reported that the 3D SPGR sequence is superior to the 2D FSE sequence in the identification of pituitary adenomas with a sensitivity of up to 80% [11,12,13], but it cannot satisfy the clinical needs that about 20% of the lesions are still missed. Therefore, techniques are needed that can help better identify pituitary adenomas, particularly microadenomas. Previously, the 3D FSE sequence was recommended in patients with hyperprolactinemia [14]. Recently, the 3D FSE sequence has developed rapidly and can provide superior image quality with diminished artifacts [15]. Sartoretti et al demonstrated in a very effective fashion that the 3D FSE sequence is a reliable alternative for pituitary imaging in terms of image quality [16]. However, to our knowledge, few studies have investigated the diagnostic performance of 3D FSE sequences for identifying ACTH-secreting pituitary adenomas, particularly microadenomas. The aim of our study was to assess the diagnostic performance of hrMRI with 3D FSE sequence by comparison with cMRI and dMRI with 2D FSE sequence for identifying ACTH-secreting pituitary microadenomas in patients with Cushing’s syndrome. Materials and methods This single-institutional retrospective study was approved by the Institutional Review Board of our hospital. The study was conducted in accordance with the Helsinki Declaration. The informed consent was waived due to the retrospective nature of the study. Study participants We retrospectively reviewed the medical records and imaging studies of 186 consecutive patients with ACTH-dependent Cushing’s syndrome, who underwent a combined protocol of cMRI, dMRI, and hrMRI from January 2016 to December 2020. Postoperative patients with Cushing’s disease (n = 97), patients with ectopic ACTH syndrome who underwent pituitary exploration (n = 2), and patients with macroadenomas (n = 5) or lack of pathology (n = 13) were excluded from the study. Finally, 69 patients with ACTH-dependent Cushing’s syndrome were included in the current study (Fig. 1) and the patients included were all surgically confirmed. Fig. 1 Flowchart of patient inclusion/exclusion process and image analysis. ACTH adrenocorticotropic hormone, CD Cushing’s disease, EAS ectopic ACTH syndrome, T1WI T1-weighted imaging, T2WI T2-weighted imaging Full size image MRI protocol All the patients were imaged on a 3.0 Tesla MR scanner (Discovery MR750w, GE Healthcare) using an 8-channel head coil. The MRI protocol included coronal T2-weighted imaging, coronal T1-weighted imaging, and sagittal T1-weighted imaging before contrast injection. After contrast injection of gadopentetate dimeglumine (Gd-DTPA) at 0.05 mmol/kg (0.1 mL/kg) with a flow rate of 2 mL/s followed by a 10-mL saline solution flush, dMRI and cMRI with 2D FSE sequence were obtained first, and hrMRI with 3D FSE sequence using variable flip angle technique was performed immediately afterward. Detailed acquisition parameters are presented in Table S1. Image analysis: diagnostic performance Image interpretation was independently conducted by two experienced neuroradiologists (F.F. and H.Y. with 25 and 16 years of experience in neuroradiology, respectively), who were blinded to patient information. The evaluation order of cMRI, dMRI, and hrMRI sequences was randomized. The identification of pituitary microadenomas on images was scored based on a three-point scale (0 = poor; 1 = fair; 2 = excellent). Scores of 1 or 2 represented the identification of the lesion. Reference standards were established by using all available imaging, clinical, surgical, and pathological resources, with a multidisciplinary team approach. Image analysis: image quality Two readers (Z.L. and B.H. with 4 years of experience in radiology, respectively) were asked to assess the image quality of cMRI, dMRI, and hrMRI. Before exposure to images used in the current study, these readers underwent a training session to make sure that they were comparable to the experienced neuroradiologists in terms of image quality assessment. Images were presented in a random order. Image quality was assessed by using a 5-point Likert scale [17], including overall image quality (1 = non-diagnostic; 2 = poor; 3 = fair; 4 = good; 5 = excellent), sharpness (1 = non-diagnostic; 2 = not sharp; 3 = a little sharp; 4 = moderately sharp; 5 = satisfyingly sharp), and structural conspicuity (1 = non-diagnostic; 2 = poor; 3 = fair; 4 = good; 5 = excellent). An example of image quality assessment is shown in Table S2. Final decision was made through a consensus agreement. The mean signal intensity of pituitary microadenomas, pituitary gland, and noise on cMRI, dMRI, and hrMRI was measured using an operator-defined region of interest. For noise, a 10-mm2 region of interest was placed in the background, and noise was defined as the standard deviation of the signal intensity of the background [17]. For pituitary microadenomas and pituitary gland, the region of interest should include a representative portion of the structure. The mean signal intensity of the pituitary microadenoma was replaced with that of the pituitary gland when no microadenoma was identified. A signal-to-noise ratio (SNR) was defined as the mean signal intensity of the pituitary microadenoma divided by noise. A contrast-to-noise ratio (CNR) was defined as the absolute difference of the mean signal intensity between the normal pituitary gland and pituitary microadenomas divided by noise [17]. Supplementary Fig. 1 shows how to measure the SNR and CNR with the region of interest in a contrast-enhanced pituitary MRI. Supplementary Fig. 2 shows the selection of images for the SNR and CNR calculation. Statistical analysis The κ analysis was conducted to assess the inter-observer agreement for identifying pituitary microadenomas. The κ value was interpreted as follows: below 0.20, slight agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, substantial agreement; greater than 0.80, almost perfect agreement. To assess the diagnostic performance of cMRI, dMRI, and hrMRI for identifying pituitary microadenomas, the receiver operating characteristic curves were plotted and the area under curves (AUCs) were compared between MR protocols for each reader by using the DeLong test. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated. The Mann–Whitney U test was used to evaluate the difference in image quality scores and the Wilcoxon signed-rank test was used to evaluate SNR and CNR measurements between MR protocols. A p value of less than 0.05 was considered statistically significant. Statistical analysis was performed using MedCalc Statistical Software (version 20.0.15; MedCalc Software) and SPSS Statistics (version 22.0; IBM). Results Clinical characteristics A total of 69 patients (median age, 39 years; interquartile range [IQR], 29–54 years; 38 women [55%]) with ACTH-dependent Cushing’s syndrome were included in the study and their clinical characteristics are shown in Table 1. Among the 69 patients, 60 (87%) patients were diagnosed with Cushing’s disease and 9 (13%) were ectopic ACTH syndrome. The median disease course was 36 months (IQR, 12–78 months). The median serum cortisol, ACTH, and 24-h urine free cortisol level before surgery were 33.0 μg/dL (IQR, 25.1–40.1 μg/dL; normal range 4.0–22.3 μg/dL), 77.2 ng/L (IQR, 55.0–124.0 ng/L; normal range 0–46 ng/L), and 422.0 μg (IQR, 325.8–984.6 μg; normal range 12.3–103.5 μg), respectively. The median serum cortisol and 24-h urine free cortisol level after surgery were 3.0 μg/dL (IQR, 1.8–18.4 μg/dL) and 195.6 μg (IQR, 63.5–1240.3 μg), respectively. The median diameter of pituitary microadenomas was 5 mm (IQR, 4–5 mm), ranging from 3 to 9 mm. Table 1 Clinical characteristics of the patients Full size table Diagnostic performance of cMRI, dMRI, and hrMRI for identifying pituitary microadenomas The inter-observer agreement for identifying pituitary microadenomas by κ statistic between two readers was moderate on cMRI (κ = 0.50), moderate on dMRI (κ = 0.57), and almost perfect on hrMRI (κ = 0.91), respectively. The diagnostic performance for identifying pituitary microadenomas on cMRI, dMRI, hrMRI, and combined cMRI and dMRI is summarized in Table 2. For reader 1, the diagnostic performance of hrMRI (AUC, 0.95; 95%CI: 0.87, 0.99) was higher than that of cMRI (AUC, 0.75; 95%CI: 0.63, 0.85; p = 0.002), dMRI (AUC, 0.59; 95%CI: 0.47, 0.71; p < 0.001), and combined cMRI and dMRI (AUC, 0.65; 95%CI: 0.53, 0.76; p = 0.001). For reader 2, the diagnostic performance of hrMRI (AUC, 0.97; 95%CI: 0.89, 1.00) was higher than that of cMRI (AUC, 0.74; 95%CI: 0.63, 0.84; p = 0.001), dMRI (AUC, 0.68; 95%CI: 0.56, 0.79; p = 0.001), and combined cMRI and dMRI (AUC, 0.70; 95%CI: 0.58, 0.80; p = 0.003). Table 2 Diagnostic performance of cMRI, dMRI, and hrMRI for identifying pituitary microadenomas Full size table For reader 1, 23 of the 69 patients (33%) were misdiagnosed on both cMRI and dMRI, but 18 of the 23 misdiagnosed patients (78%) were correctly diagnosed on hrMRI. For reader 2, 17 of the 69 patients (25%) were misdiagnosed on both cMRI and dMRI, but 14 of the 17 misdiagnosed patients (82%) were correctly diagnosed on hrMRI. Figure 2 shows that a 5-mm pituitary microadenoma was identified on preoperative pituitary MRI. The margin of the lesion was fully delineated on hrMRI, but not on cMRI and dMRI. Figure 3 shows that a 3-mm pituitary microadenoma was missed on cMRI, but identified on dMRI and hrMRI. Figure 4 shows that a 5-mm pituitary microadenoma was correctly diagnosed on hrMRI, but missed on cMRI or dMRI. Figure 5 shows that a 4-mm pituitary microadenoma was evident on coronal images as well as reconstructed axial and reconstructed sagittal images on hrMRI. Fig. 2 Images in a 56-year-old man with Cushing’s disease. The 5-mm pituitary microadenoma (arrow) can be identified on (a) coronal contrast-enhanced T1-weighted image and (b) coronal dynamic contrast-enhanced T1-weighted image obtained with two-dimensional (2D) fast spin echo (FSE) sequence, but the margin is not fully delineated. The lesion (arrow) is well delineated on (c) coronal contrast-enhanced T1-weighted image on high-resolution MRI obtained with 3D FSE sequence. d Intraoperative endoscopic photograph during transsphenoidal surgery after exposure of the sellar floor shows a round pituitary microadenoma (arrow) Full size image Fig. 3 Images in a 34-year-old woman with Cushing’s disease. No tumor is identified on (a) coronal contrast-enhanced T1-weighted image obtained with two-dimensional (2D) fast spin echo (FSE) sequence. The 3-mm pituitary microadenoma (arrow) with delayed enhancement is identified on the left side of the pituitary gland on (b) coronal dynamic contrast-enhanced T1-weighted image obtained with 2D FSE sequence and (c) coronal contrast-enhanced T1-weighted image on high-resolution MRI obtained with 3D FSE sequence. d Intraoperative endoscopic photograph during transsphenoidal surgery shows a 3-mm pituitary microadenoma (arrow) Full size image Fig. 4 Images in a 43-year-old man with Cushing’s disease. The lesion is missed on (a) coronal contrast-enhanced T1-weighted image and (b) coronal dynamic contrast-enhanced T1-weighted image obtained with two-dimensional (2D) fast spin echo (FSE) sequence. c Coronal contrast-enhanced T1-weighted image on high-resolution MRI obtained with 3D FSE sequence shows a round pituitary microadenoma (arrow) measuring approximately 5 mm with delayed enhancement on the left side of the pituitary gland. d Intraoperative endoscopic photograph for microsurgical resection of the 5-mm pituitary microadenoma (arrow) Full size image Fig. 5 Images in a 48-year-old woman with Cushing’s disease. Preoperative high-resolution contrast-enhanced MRI using three-dimensional fast spin echo sequence shows a 4-mm pituitary microadenoma (arrow) with delayed enhancement is well delineated on the left side of the pituitary gland on (a) coronal, (b) reconstructed axial, and (c) reconstructed sagittal contrast-enhanced T1-weighted images. d Intraoperative endoscopic photograph during transsphenoidal surgery after exposure of the sellar floor shows a round pituitary microadenoma (arrow) Full size image Image quality of cMRI, dMRI, and hrMRI Image quality scores of cMRI, dMRI, and hrMRI are presented in Table 3. Scores for overall image quality, sharpness, and structural conspicuity on hrMRI (overall image quality, 5.0 [IQR, 5.0–5.0]; sharpness, 5.0 [IQR, 4.5–5.0]; structural conspicuity, 5.0 [IQR, 5.0–5.0]) were higher than those on cMRI (overall image quality, 4.0 [IQR, 3.5–4.0]; sharpness, 4.0 [IQR, 3.0–4.0]; structural conspicuity, 4.0 [IQR, 4.0–4.0]; p < 0.001 for all) and dMRI (overall image quality, 4.0 [IQR, 4.0–4.0]; sharpness, 4.0 [IQR, 4.0–4.0]; structural conspicuity, 4.0 [IQR, 4.0–4.5]; p < 0.001 for all). Table 3 Image quality scores on cMRI, dMRI, and hrMRI Full size table The SNR and CNR measurements are shown in Table 4. The SNR of the pituitary microadenomas on hrMRI (67.5 [IQR, 51.2–92.1]) was lower than that on cMRI (82.3 [IQR, 61.8–127.2], p < 0.001), but higher than that on dMRI (53.9 [IQR, 35.2–72.6], p = 0.001). The CNR on hrMRI (26.2 [IQR, 15.1–41.0]) was higher than that on cMRI (10.6 [IQR, 0–42.6], p = 0.023) and dMRI (11.2 [IQR, 0–29.8], p < 0.001). Table 4 SNR and CNR on cMRI, dMRI, and hrMRI Full size table Discussion The identification of pituitary microadenomas is considerably challenging but critical in patients with ACTH-dependent Cushing’s syndrome. Our study demonstrated that hrMRI with 3D FSE sequence had higher diagnostic performance (AUC, 0.95–0.97) than cMRI (AUC, 0.74–0.75; p ≤ 0.002) and dMRI (AUC, 0.59–0.68; p ≤ 0.001) for identifying pituitary microadenomas. To our knowledge, there are no previous studies specifically evaluating the identification of pituitary microadenomas on hrMRI with 3D FSE sequence by comparison with cMRI and dMRI in patients with ACTH-dependent Cushing’s syndrome, and this is the largest study conducted in ACTH-secreting microadenomas with a sensitivity of more than 90%. Recently, techniques for pituitary evaluation have developed rapidly. Because of false negatives and false positives on cMRI and dMRI using 2D FSE sequence [7, 9, 10], a 3D SPGR sequence was introduced for identifying pituitary adenomas. Previous studies demonstrated that the 3D SPGR sequence performed better than the 2D FSE sequence in the identification of pituitary adenomas with a sensitivity of up to 80% [11,12,13]. In patients with hyperprolactinemia, the 3D FSE sequence was recommended [14] and the 3D FSE sequence has rapidly developed recently with superior image quality [15, 16], suggesting that the 3D FSE sequence may be a reliable alternative for identifying pituitary adenomas. However, to our knowledge, few studies have investigated the diagnostic performance of the 3D FSE sequence for identifying ACTH-secreting pituitary adenomas. To fill the gaps, we conducted the current study and revealed that images obtained with the 3D FSE sequence had higher sensitivity (90–93%) in identifying pituitary microadenomas, than that in previous studies using the 3D SPGR sequence [8, 11,12,13]. There is a trade-off between spatial resolution and image noise. The reduced slice thickness can overcome the partial volume averaging effect, but it is associated with increased image noise [17]. Strikingly, our study showed that hrMRI had higher image quality scores than cMRI and dMRI, in terms of overall image quality, sharpness, and structural conspicuity. The SNR of the pituitary microadenomas on cMRI was slightly higher than that on hrMRI in our study. This is because the SNR was calculated as the mean signal intensity of the pituitary gland (instead of the pituitary microadenoma) divided by noise when no microadenoma was identified, and the mean signal intensity of the pituitary gland is higher than that of the pituitary microadenoma. About 40% of pituitary microadenomas were missed on cMRI, whereas less than 10% of pituitary microadenomas were missed on hrMRI. Given the situation mentioned above, the SNR on hrMRI was lower than that on cMRI. However, the CNR on hrMRI was significantly higher than that on cMRI and dMRI. Therefore, hrMRI in our study can dramatically improve the spatial resolution with high CNR, enabling the better identification of pituitary microadenomas. The identification of pituitary adenomas on preoperative MRI in patients with ACTH-dependent Cushing’s syndrome could help the differential diagnosis of Cushing’s syndrome and aids surgical resection of lesions. It should be noted that most of the pituitary adenomas in patients with Cushing’s disease are microadenomas [5, 6]. In our study, all the tumors are microadenomas with a median diameter of 5 mm (IQR, 4–5 mm), making the diagnosis more challenging. The sensitivity of identifying pituitary adenomas decreased from 80 to 72% after excluding macroadenomas in a previous study [12], whereas the sensitivity of identifying pituitary microadenomas in our study was 90–93% on hrMRI. In the current study, hrMRI performed better than cMRI, dMRI, and combined cMRI and dMRI, with high AUC (0.95–0.97), high sensitivity (90–93%), and high specificity (100%), superior to previous studies [8, 11,12,13]. The high sensitivity of hrMRI for identifying pituitary adenomas will help surgeons improve the postoperative remission rate [4]. The high specificity of hrMRI will assist clinicians to consider ectopic ACTH syndrome, and then perform imaging to identify ectopic tumors. Besides, the inter-observer agreement for identifying pituitary microadenomas was almost perfect on hrMRI (κ = 0.91), which was moderate on cMRI (κ = 0.50) and dMRI (κ = 0.57). Therefore, hrMRI using the 3D FSE sequence is a potential alternative that can significantly improve the identification of pituitary microadenomas. Limitations of the study included its retrospective nature and the relatively small sample size in patients with ectopic ACTH syndrome as negative controls. The bias may be introduced in the patient inclusion process. Only those patients who underwent all the cMRI, dMRI, and hrMRI scans were included. In fact, some patients will bypass hrMRI when obvious pituitary adenomas were detected on cMRI and dMRI. These patients were not included in the current study because of lack of hrMRI findings. Given the situation, the sensitivity of identifying pituitary adenomas will be higher with the enrollment of these patients. Besides, the timing of the sequence acquisition after contrast injection is essential [16] and bias may be introduced due to the postcontrast enhancement curve of both the pituitary gland and the microadenoma [14]. In the future, a prospective study with different sequence acquisition orders is needed to minimize possible interference caused by the postcontrast enhancement curve. Moreover, a larger sample size is also needed to verify the diagnostic performance of hrMRI using 3D FSE sequence for identifying pituitary microadenomas and to determine whether it can replace 2D FSE or 3D SPGR sequences for routinely evaluating the pituitary gland. In conclusion, hrMRI with 3D FSE sequence showed higher diagnostic performance than cMRI and dMRI for identifying pituitary microadenomas in patients with Cushing’s syndrome. Abbreviations ACTH: Adrenocorticotropic hormone AUC: Area under the receiver operating characteristics curve cMRI: Conventional contrast-enhanced MRI CNR: Contrast-to-noise ratio dMRI: Dynamic contrast-enhanced MRI FSE: Fast spin echo hrMRI: High-resolution contrast-enhanced MRI IQR: Interquartile range SNR: Signal-to-noise ratio SPGR: Spoiled gradient re called References Lacroix A, Feelders RA, Stratakis CA, Nieman LK (2015) Cushing’s syndrome. Lancet 386:913–927 Article CAS PubMed Google Scholar Loriaux DL (2017) Diagnosis and differential diagnosis of Cushing’s syndrome. N Engl J Med 376:1451–1459 Article CAS PubMed Google Scholar Nieman LK, Biller BM, Findling JW et al (2015) Treatment of Cushing’s syndrome: an Endocrine Society clinical practice guideline. 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Funding This study has received funding from the National Natural Science Foundation of China (grant 82071899), the National Key Research and Development Program of China (grants 2016YFC1305901, 2020YFA0804500), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (grants 2017-I2M-3–008, 2021-I2M-1–025), the Beijing Natural Science Foundation (grant L182067) and National High Level Hospital Clinical Research Funding (2022-PUMCH-B-067, 2022-PUMCH-B-114). Author information Author notes Zeyu Liu and Bo Hou contributed equally to this work and share first authorship Hui You and Feng Feng contributed equally to this work and share corresponding authorship Authors and Affiliations Department of Radiology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Shuaifuyuan Wangfujing Dongcheng Distinct, Beijing, 100730, China Zeyu Liu, Bo Hou, Hui You, Mingli Li & Feng Feng Department of Endocrinology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Shuaifuyuan Wangfujing Dongcheng Distinct, Beijing, 100730, China Lin Lu, Lian Duan & Huijuan Zhu Department of Neurosurgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Shuaifuyuan Wangfujing Dongcheng Distinct, Beijing, 100730, China Kan Deng & Yong Yao State Key Laboratory of Complex Severe and Rare Disease, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Shuaifuyuan Wangfujing Dongcheng Distinct, Beijing, 100730, China Yong Yao, Huijuan Zhu & Feng Feng Corresponding authors Correspondence to Hui You or Feng Feng. Ethics declarations Guarantor The scientific guarantor of this publication is Feng Feng. Conflict of interest The authors of this manuscript declare no conflict of interest. Statistics and biometry No complex statistical methods were necessary for this paper. Informed consent Written informed consent was waived by the Institutional Review Board. Ethical approval Institutional Review Board approval was obtained. Methodology • retrospective • diagnostic or prognostic study • performed at one institution Additional information Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. 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From Janice: A devastating horrific disease that doctors won’t believe we have. Feeling like we will collapse with each step. Carrying an empty plastic bag seems too heavy to manage. And no one will believe us. They think we are lazy, fat and crazy. What is your experience?

Abstract Importance Cushing syndrome is defined as a prolonged increase in plasma cortisol levels that is not due to a physiological etiology. Although the most frequent cause of Cushing syndrome is exogenous steroid use, the estimated incidence of Cushing syndrome due to endogenous overproduction of cortisol ranges from 2 to 8 per million people annually. Cushing syndrome is associated with hyperglycemia, protein catabolism, immunosuppression, hypertension, weight gain, neurocognitive changes, and mood disorders. Observations Cushing syndrome characteristically presents with skin changes such as facial plethora, easy bruising, and purple striae and with metabolic manifestations such as hyperglycemia, hypertension, and excess fat deposition in the face, back of the neck, and visceral organs. Cushing disease, in which corticotropin excess is produced by a benign pituitary tumor, occurs in approximately 60% to 70% of patients with Cushing syndrome due to endogenous cortisol production. Evaluation of patients with possible Cushing syndrome begins with ruling out exogenous steroid use. Screening for elevated cortisol is performed with a 24-hour urinary free cortisol test or late-night salivary cortisol test or by evaluating whether cortisol is suppressed the morning after an evening dexamethasone dose. Plasma corticotropin levels can help distinguish between adrenal causes of hypercortisolism (suppressed corticotropin) and corticotropin-dependent forms of hypercortisolism (midnormal to elevated corticotropin levels). Pituitary magnetic resonance imaging, bilateral inferior petrosal sinus sampling, and adrenal or whole-body imaging can help identify tumor sources of hypercortisolism. Management of Cushing syndrome begins with surgery to remove the source of excess endogenous cortisol production followed by medication that includes adrenal steroidogenesis inhibitors, pituitary-targeted drugs, or glucocorticoid receptor blockers. For patients not responsive to surgery and medication, radiation therapy and bilateral adrenalectomy may be appropriate. Conclusions and Relevance The incidence of Cushing syndrome due to endogenous overproduction of cortisol is 2 to 8 people per million annually. First-line therapy for Cushing syndrome due to endogenous overproduction of cortisol is surgery to remove the causative tumor. Many patients will require additional treatment with medications, radiation, or bilateral adrenalectomy. From https://jamanetwork.com/journals/jama/article-abstract/2807073

In this application note, Tecan presents a method for diagnosing Cushing's syndrome efficiently and accurately. The approach involves simultaneous the measurement of cortisol and dexamethasone levels using LC-MS/MS, which reduces false positives in dexamethasone suppression test (DSTs). The described LC-MS/MS method enables the tracking of multiple analytes, including cortisol, cortisone, and dexamethasone, in serum or plasma. Implementing this analytical approach offers clinical laboratories a straightforward means of performing DSTs, and the availability of a commercially available kit ensures reliable and reproducible results. Download this Article Download PDF >> From https://www.selectscience.net/application-articles/minimizing-the-number-of-false-positives-in-dexamethasone-suppression-testing-for-the-diagnosis-of-cushings-syndrome?artID=59632

Abstract Rationale: Ectopic ACTH-producing pituitary adenoma (EAPA) of the clivus region is extraordinarily infrequent condition and merely a few reports have been reported to date. Patient concerns: The patient was a 53-year-old woman who presented with Cushing-like appearances and a soft tissue mass in the clivus region. Diagnoses: The final diagnosis of clivus region EAPA was established by clinical, radiological and histopathological findings. Interventions: The patient underwent gross total clivus tumor resection via transsphenoidal endoscopy. Outcomes: Half a year after surgery, the patient Cushing-like clinical manifestations improved significantly, and urinary free cortisol and serum adrenocorticotropin (ACTH) returned to normal. Lessons: Given the extreme scarcity of these tumors and their unique clinical presentations, it may be possible to misdiagnose and delayed treatment. Accordingly, it is especially crucial to summarize such lesions through our present case and review the literature for their precise diagnosis and the selection of optimal treatment strategies. 1. Introduction Pituitary adenoma arises from the anterior pituitary cells and is the commonest tumor of the sellar region.[1] It makes up approximately 10% to 15% of all intracranial tumors.[2] Ectopic pituitary adenoma (EPA) is defined as a pituitary adenoma that occurs outside the sellar area and has no direct connection to normal pituitary tissue.[3] The most frequent sites of EPA are the sphenoid sinus and suprasellar region, and much less frequent sites including the clivus region, cavernous sinus, and nasopharynx.[4] Hypercortisolism and the series of symptoms it leads to is termed Cushing syndrome (CS).[5] CS is classified into adrenocorticotropin (ACTH)-dependent and ACTH-independent CS depending on the cause, accounting for 80% to 85% and 15% to 20% of cases, respectively.[6] Pituitary adenoma accounts for ACTH-dependent CS 75% to 80%, while ectopic ACTH secretion accounts for the remaining 15% to 20%.[7] Ectopic CS is a very rare disorder of CS caused by an ACTH-secreting tumor outside the pituitary or adrenal gland.[8] It has been reported that ectopic ACTH-producing pituitary adenoma (EAPA) can occur in the sphenoid sinus, cavernous sinus, clivus, and suprasellar region,[9] with EAPA in the clivus region being extremely rare, and merely 6 cases have been reported in the English literature (Table 1).[10–15] Furthermore, as summarized in the Table 1, EAPA in the clivus area has unique symptoms, which may lead to misdiagnosis as well as delay in treatment. Therefore, we herein described a case of CS from an EAPA of the clivus region and reviewed relevant literature for the purpose of further understanding this extraordinarily unusual condition. Table 1 - Literature review of cases of primary clival ectopic ACTH-producing pituitary adenoma (including the current case). Reference Age (yr)/sex Symptoms Imaging findings Maximum tumor diameter (mm) Preoperative elevated hormone IHC Surgery RT Follow-up (mo) Outcome Ortiz et al 1975[10] 15/F NA NA NA NA NA Right transfrontal craniotomy, NA Yes NA Symptomatic relief Anand et al 1993[11] 58/F Anosphrasia, blurred vision, occasional left frontal headache, Routine radiographic evaluation revealed a clival tumor and nasopharyngeal mass with bone erosion. MRI demonstrated a Midline homogeneous mass. 30 ACTH ACTH in a few isolated cells Maxillotomy approach, GTR Yes 12 Symptomatic relief Pluta et al 1999[12] 20/F Cushing syndrome MRI revealed a hypodense contrast-enhancing lesion. NA ACTH ACTH Transsphenoidal surgery, GTR No 18 Symptomatic relief Shah et al 2011[13] 64/M Facial paresthesias, myalgias, decreased muscle strength, and fatigue CT imaging showed a clival mass. 21 ACTH ACTH NA, GTR No 7 Symptomatic relief Aftab et al 2021[14] 62/F Transient unilateral visual loss MRI showed a T2 heterogeneously enhancing hyperintense lesion. 21 No ACTH Transsphenoidal resection, GTR NO 6 Symptomatic relief Li et al 2023[15] 47/F Bloody nasal discharge, dizziness and headache CT revealed an ill-defined mass eroding the adjacent bone. MRI T1 showed a heterogeneous mass with hypointensity, hyperintensity on T2-weighted images and isointensity on diffusion-weighted images. 58 NA ACTH Transsphenoidal endoscopy, STR Yes 2 Symptomatic relief Current case 53/F Headache, and dizziness, Cushing syndrome CT demonstrated bone destruction and a soft tissue mass. MRI T1 revealed irregular isointense signal, and MRI T2 showed isointense signal/slightly high signal. 46 ACTH ACTH Transsphenoidal endoscopy, GTR NO 6 Symptomatic relief ACTH = adrenocorticotropin, CT = computed tomography, GTR = gross total resection, IHC = immunohistochemistry, MRI = magnetic resonance imaging, NA = not available, RT = radiotherapy, STR = subtotal resection. 2. Case presentation A 53-year-old female presented to endocrinology clinic of our hospital with headache and dizziness for 2 years and aggravated for 1 week. Her past medical history was hypertension, with blood pressure as high as 180/100 mm Hg. Her antihypertensive medications included amlodipine besylate, benazepril hydrochloride, and metoprolol tartrate, and she felt her blood pressure was well controlled. In addition, she suffered a fracture of the thoracic vertebrae 3 month ago; and bilateral rib fractures 1 month ago. Physical examination revealed that the patient presented classical Cushing-like appearances, including moon face and supraclavicular and back fat pads, and centripetal obesity (body mass index, 25.54 kg/m2) with hypertension (blood pressure, 160/85 mm Hg). Laboratory studies revealed high urinary free cortisol levels at 962.16 µg/24 hours (reference range, 50–437 µg/24 hours) and absence of circadian cortisol rhythm (F [0am] 33.14 µg/dL, F [8am] 33.52 µg/dL, F [4pm] 33.3 µg/dL). ACTH levels were elevated at 90.8 pg/mL (reference range, <46 pg/mL). The patient low-dose dexamethasone suppression test demonstrated the existence of endogenous hypercortisolism. High-dose dexamethasone suppression test results revealed that serum cortisol levels were suppressed by <50%, suggesting the possibility of ectopic ACTH-dependent CS. Serum luteinizing hormone and serum follicle stimulating hormone were at low levels, <0.07 IU/L (reference range, 15.9–54.0 IU/L) and 2.57 IU/L (reference range, 23.0–116.3 IU/L), respectively. Insulin-like growth factor-1, growth hormone (GH), prolactin (PRL), thyroid stimulating hormone, testosterone, progesterone and estradiol test results are all normal. Oral glucose tolerance test showed fasting glucose of 6.3 mmol/L and 2-hour glucose of 18.72 mmol/L; glycosylated hemoglobin (HbA1c) was 7.1%. Serum potassium fluctuated in the range of 3.14 to 3.38 mmol/L (reference range, 3.5–5.5 mmol/L), indicating mild hypokalemia. High-resolution computed tomography (CT) scan of the sinuses revealed osteolytic bone destruction of the occipital clivus and a soft tissue mass measuring 20 mm × 30 mm × 46 mm (Fig. 1A). The mass filled the bilateral sphenoid sinuses and involved the cavernous sinuses, but the pituitary was normal. Cranial MR scan showed the T1W1 isointense signal and the T2W1 isointense signal/slightly high signal in the sphenoid sinus and saddle area (Fig. 1B–D). Bone density test indicated osteoporosis. Figure 1.: Radiological findings. (A) CT demonstrated bone destruction and a soft tissue mass on the occipital clivus (white arrow). (B) Axial view of the MR T1 revealed irregular isointense signal in the sphenoid sinus and saddle area (white arrow). (C and D) Axial view and sagittal view of the MR T2 showed isointense signal/slightly high signal in the sphenoid sinus and saddle area (black arrow). CT = computed tomography. Subsequently, the patient underwent gross total clivus tumor resection via transsphenoidal endoscopy. During surgery, the tumor was found to be light red in color with a medium texture, and the tumor tissue protruded into the sphenoidal sinus cavity and eroded the clival area. Histologically, the tumor cells were nested, with interstitially rich blood sinuses and organoid arrangement (Fig. 2A). The tumor cells were relatively uniform in size, with light red cytoplasm, delicate pepper salt-like chromatin, and visible nucleoli (Fig. 2B). In addition, mitosis of tumor cells was extremely rare. Immunohistochemically, the neoplasm cells were diffuse positive for CK (Fig. 2C), CgA (Fig. 2D), ACTH (Fig. 2E), Syn and CAM5.2, with low Ki-67 labeling index (<1%) (Fig. 2F). Simultaneously, all other pituitary hormone markers like GH, thyroid stimulating hormone, PRL, luteinizing hormone, as well as follicle stimulating hormone were negatively expressed. On the basis of these medically historical, clinical, laboratorial, morphologic, and immunohistochemical findings, the final pathological diagnosis of an EAPA was established. Figure 2.: HE and immunohistochemical findings. (A) Histologic sections revealed morphologically homogeneous tumor cells in nests with a prominent and delicate vascularized stroma (H&E, × 200). (B) The tumor cells had fine chromatin with visible nuclei and rare mitoses (H&E, × 400). CK (C), CgA (D) and ACTH (E) immunohistochemically showed diffuse reactivity of the tumor cells (SP × 200). (F) The proliferation index is <1% on Ki-67 staining (SP × 200). When evaluated 2 months after surgery, her Cushing-like characteristics had well improved, and her blood pressure was normal. Furthermore, her serum cortisol and ACTH returned to the normal levels. Six-month postoperative follow-up revealed that serum cortisol and ACTH were stable at normal levels, and no signs of tumor recurrence were detected on imaging. 3. Discussion EAPA is defined as an ACTH-secreting ectopic adenoma located outside the ventricles, and has no continuity with the normal intrasellar pituitary gland.[9] ACTH promotes cortisol secretion by stimulating the adrenal cortical fasciculus. The clinical manifestations of hypercortisolism are diverse, and the severity is partly related to the duration of the cortisol increase.[8] Clival tumors are typically uncommon, accounting for 1% of all intracranial tumors. There are many differential diagnoses for clival lesions, including the most common chordoma (40%), meningioma, chondrosarcoma, astrocytoma, craniopharyngioma, germ cell tumors, non-Hodgkin lymphoma, melanoma, metastatic carcinoma, and rarely pituitary adenoma.[16] The commonest clival EPA is a PRL adenoma, followed by null cell adenoma, and the least common are ACTH adenoma and GH adenoma.[2] The clival EAPA is extremely unwonted, and only 6 other cases apart from ours have been reported in literature so far (Table 1). The average age of the patients with these tumors was 48 years (range, 15–64 years). There was a obvious female predominance with a female-to-male prevalence ratio of 6:1. Only 2 patients (2/6, 33.3%) with reported clinical symptoms, including our patients, presented with overt clinical manifestations of CS. Compression of the mass on adjacent structures (e.g., nerves) may result in anosphrasia, visual impairment, headache, myalgias, decreased muscle strength, dizziness and facial sensory abnormalities. The diagnosis and localization of these tumors relied heavily on radiological imaging. Head MRI was the most basic method used for them detection, for localization adenomas and their invasion of surrounding structures to guide the choice of treatment and surgical options methods. Radiographic characteristics had been reported in 6 patients with EAPA in the clivus region. All of these patients (6/6, 100%) had initial positive findings of sellar MRI (or CT) identifying an ectopic adenoma before surgery. MR T1 was usually a low-intensity or isointense signal, while MR T2 was usually an isointense or slightly higher signal. The maximum diameter of the tumor was reported in 5 cases, with the mean maximum diameter was 35.2 mm (range, 21–55 mm) according to preoperative MRI and intraoperative observations. As summarized in Table 1, 4/5 clival EAPA cases secreted ACTH. Histologically, all cases (6/6, 100%) expressed ACTH scatteredly or diffusely. The gold standard for the treatment of CS caused by EAPA was the surgical removal of EPA, which was essential to achieve remission and histological confirmation of the disease.[9] The most common method of EAPA resection in the clivus region was transsphenoidal sinus resection (4/6, 66.67%), followed by craniotomy (1/6, 16.67%) and maxillary osteotomy (1/6, 16.67%). Transsphenoidal endoscopic surgery allowed resection of the EAPA and manipulation of neurovascular structures and avoidance of cerebral atrophy, whereas craniotomy allowed full exposure of the suprasellar region, direct visualization or manipulation of the adenoma, and reduced the risk of postoperative CSF leak.[9] Both approaches had their advantages, and there was no consensus on which surgical approach was best for the treatment of EAPA in the slope area.[9] The choice of the best surgical approach was believed to be based on the condition of the adenoma, as well as the general condition of the patient and the experience of the surgeon.[9] As summarized in Table 1, most complete tumor resections were achieved regardless of the method chosen. A minority of patients underwent postoperative radiotherapy (3/7, 42.86%), and most of them had invasion of the surrounding bone tissue. All patients experienced effective postoperative relief of symptoms. In summary, due to the rarity of this disorder, an accurate preoperative diagnosis of EAPA in the slope area is extremely challenging for the clinician or radiologist. The final precise diagnosis relies on a combination of clinical symptoms, imaging findings, histology and immunohistochemical markers. For this type of tumor, surgery is an effective treatment to relieve the clinical manifestations caused by tumor compression or hormonal secretion. The choice of postoperative adjuvant radiotherapy is mainly based on the presence of invasion of the surrounding bone tissue. Further cases may be necessary to summarize the clinical features of such lesions and to develop optimal treatment strategies. Acknowledgments We would like to thank the patient and her family. Author contributions Conceptualization: Yutao He. Data curation: Ziyi Tang. Formal analysis: Na Tang. Methodology: Yu Lu, Fangfang Niu, Jiao Ye, Zheng Zhang, Chenghong Fang. Writing – original draft: Yutao He. Writing – review & editing: Yutao He, Lei Yao. Abbreviations: ACTH adrenocorticotropin CS cushing syndrome CT computed tomography EAPA ectopic ACTH-producing pituitary adenoma EPA ectopic pituitary adenoma GH growth hormone PRL prolactin References [1]. Gittleman H, Ostrom QT, Farah PD, et al. Descriptive epidemiology of pituitary tumors in the United States, 2004-2009. J Neurosurg. 2014;121:527–35. Cited Here | PubMed | CrossRef | Google Scholar [2]. Karras CL, Abecassis IJ, Abecassis ZA, et al. Clival ectopic pituitary adenoma mimicking a Chordoma: case report and review of the literature. Case Rep Neurol Med. 2016;2016:8371697. Cited Here | Google Scholar [3]. Bălaşa AF, Chinezu R, Teleanu DM, et al. Ectopic intracavernous corticotroph microadenoma: case report of an extremely rare pathology. Rom J Morphol Embryol. 2017;58:1447–51. Cited Here | Google Scholar [4]. Zhu J, Wang Z, Zhang Y, et al. Ectopic pituitary adenomas: clinical features, diagnostic challenges and management. 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Cushing syndrome caused by ectopic adrenocorticotropic hormone-secreting pituitary adenomas: case report and literature review. World Neurosurg. 2020;142:75–86. Cited Here | Google Scholar [10]. Ortiz-Suarez H, Erickson DL. Pituitary adenomas of adolescents. J Neurosurg. 1975;43:437–9. Cited Here | PubMed | CrossRef | Google Scholar [11]. Anand VK, Osborne CM, Harkey HL. Infiltrative clival pituitary adenoma of ectopic origin. Otolaryngol Head Neck Surg. 1993;108:178–83. Cited Here | PubMed | CrossRef | Google Scholar [12]. Pluta RM, Nieman L, Doppman JL, et al. Extrapituitary parasellar microadenoma in Cushing’s disease. J Clin Endocrinol Metab. 1999;84:2912–23. Cited Here | View Full Text | PubMed | CrossRef | Google Scholar [13]. Shah R, Schniederjan M, DelGaudio JM, et al. Visual vignette.s Ectopic ACTH-secreting pituitary adenoma. Endocr Pract. 2011;17:966. Cited Here | Google Scholar [14]. Aftab HB, Gunay C, Dermesropian R, et al. “An Unexpected Pit” - ectopic pituitary adenoma. J Endocr Soc. 2021;5:A557–8. Cited Here | Google Scholar [15]. Li Y, Zhu JG, Li QQ, et al. Ectopic invasive ACTH-secreting pituitary adenoma mimicking chordoma: a case report and literature review. BMC Neurol. 2023;23:81. Cited Here | Google Scholar [16]. Wong K, Raisanen J, Taylor SL, et al. Pituitary adenoma as an unsuspected clival tumor. Am J Surg Pathol. 1995;19:900–3. Cited Here | View Full Text | PubMed | CrossRef | Google Scholar Hide full references list Keywords: clivus region; Cushing; Ectopic ACTH; like appearance; producing pituitary adenoma From https://journals.lww.com/md-journal/Fulltext/2023/06230/Cushing_syndrome_caused_by_an_ectopic.32.aspx

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In Italy it is estimated that there are about 3,000 patients suffering from Cushing’s syndrome, while in Europe the number rises to over 50,000. The Cushing’s syndrome, a disease caused by the excessive production of cortisol by the pituitary gland due to a benign tumor of the gland, has seen a breakthrough in its treatment. Thanks to a new drug called osilodrostat, approved in 2020 by the Food and Drug Administration and subsequently by Aifa in Italy, patients unfit for surgery can benefit from a treatment that offers the same effects as a scalpel. Furthermore, this drug reduced symptoms in 80% of cases. Cushing’s syndrome has been dubbed “full moon face disease” due to its most obvious visible effects, such as a rounding of the face caused by fat accumulation and visible weight gain also on the waist and back. Despite its symptomatic relevance, the disease has long been poorly understood by both healthcare professionals and the general public. To raise awareness of this syndrome, the #Thiscushing campaign has been launched, which aims to spread knowledge about the disease. The campaign recently stopped in Rome, during the Congress of the Italian Society of Endocrinology (SIE), where a photographic exhibition was organized which represents moments of daily life of people affected by Cushing’s syndrome and their difficulties. Despite the debilitating symptoms, Cushing’s syndrome is often underdiagnosed, resulting in delays in diagnosis of up to 5-7 years. The disease presents a wide range of symptoms, ranging from difficulty performing even simple daily activities such as tying your shoes or getting out of bed, to common manifestations such as high cholesterol, hypertension and hyperglycemia, which can be confused with symptoms of other less common pathologies. serious. It is for this reason that the EIS experts are appealing for the inclusion of Cushing’s syndrome in the list of rare pathologies recognized by the Ministry of Health, in order to facilitate timely diagnosis and faster access to the necessary treatments. From https://www.breakinglatest.news/health/cushings-syndrome-a-new-drug-allows-you-to-avoid-surgery/